Process to produce organic compounds from synthesis gases

ABSTRACT

At least one isolated microorganism and a fermentation method to convert hydrogen gas, carbon dioxide gas, and/or carbon monoxide gas to a lower alkyl alcohol and/or carboxylic acid and to produce at least 2% by volume of the lower alkyl alcohol or carboxylic acid in an aqueous-based medium.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 13/430,203, filed Mar. 26, 2012, and it claimspriority to U.S. Pat. No. 8,178,329, filed Sep. 27, 2011, which claimspriority to PCT Application No. PCT/US2010/029707 filed, Apr. 1, 2010,which claims priority to U.S. Provisional Patent Application No.61/165,654 filed, Apr. 1, 2009.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process for producing organiccompounds such as lower alkyl alcohols, including ethanol, propanol(e.g. 1-propanol, iso-propanol), and butanol (e.g. 1-butanol), fromgases including carbon dioxide, carbon monoxide, and hydrogen underthermodynamically favorable conditions; microorganisms used in theprocess to produce organic compounds from gases; and a process forenriching, isolating, and improving microorganisms that can be used inthe process to produce organic compounds from gases. The process mayalso be used to produce one or more carboxylic acids including aceticacid, propionic acid, or butyric acid, other carboxylic acids,especially longer carboxylic acids, and the process produces animalfeeds, and can be used to produce other products.

2. Description of the Background

Currently most fuel ethanol produced in the U.S. is made from corngrain. Even if all the corn grain produced in the US were converted toethanol, it would only supply about 15% of our current transportationfuel needs. Thus, there is a pressing need to produce fuel ethanol andother alcohols from other sources of feedstock. If ethanol could beinexpensively produced from plant fiber, waste biomass like leaves,paper, manure, wood byproducts, and others materials, it could offsetfuel shortages. Plant fiber, also called cellulosic biomass, can begrown on marginal land and in greater yields than grain crops.Eventually, the U.S. aims to use up to a billion tons of such biomassper year. Other waste biomass includes garbage comprised of wasteplastic or other forms of fossil fuel derivatives.

Plant fiber is also called plant cell wall, which is comprised ofcellulose, hemicellulose, pectin, and lignin. There are a few processesavailable for the production of ethanol from plant fiber. One process isphysical conversion: biomass is heated to high temperatures, such as650° F. The biomass is degraded to carbon monoxide (CO) and hydrogen(H₂), and subsequently these gases are converted to ethanol by acatalytic or microbial process. The advantage of this approach is thatmany forms of biomass or fossil fuel derivatives can be used, but thecost of facilities may be high compared to anaerobic digestion. Inaddition, waste gases from other industrial processes can be used, oreven gases produced by anaerobic digestion can be efficiently used.

Use of microorganisms to produce acetic acid or ethanol from CO₂, CO andH₂ was disclosed in U.S. Pat. No. 5,173,429; U.S. Pat. No. 5,593,886;and U.S. Pat. No. 6,136,577, which are incorporated herein by reference.However, the ratio of acetic acid to ethanol was 20:1 or greater andonly 0.1% ethanol concentration could be achieved. In U.S. Pat. No.7,285,402, incorporated herein by reference, ethanol concentrationsgreater than 10 g/L and acetate concentrations lower than about 8-10 g/Lwere claimed, while continuing to permit culture growth and good culturestability. However, the cost of achieving these rates through physicalmanipulations of the fermentation, and the cost of distillation for suchlow concentrations of ethanol would be cost prohibitive for anindustrial process.

A second approach is called biochemical conversion: the biomass isboiled in caustic acids or other chemicals to hydrolyze the celluloseand hemicellulose. The residue is neutralized and conditioned andsubjected to cellulolytic enzymes to release sugars. The glucosereleased is fermented by yeast to ethanol, and the 5-carbon sugars areseparated and converted to ethanol by a separate organism.

A third approach to producing cellulosic ethanol would be to use livingmicroorganisms that can digest cellulose, hemicellulose and pectins andconvert them to ethanol. This approach would be least expensive becauseit does not require harsh chemicals or high temperatures and uses fewerprocessing steps. However, the approach is only feasible if there is amicroorganism, or mixed culture of microorganisms, that can readilydigest cellulose and hemicellulose, and which, preferably converts asignificant part of the carbohydrate to ethanol. The ideal organismswould also be tolerant to ethanol concentrations so that they can beused to digest considerable carbohydrate to ethanol at high enoughconcentration to decrease the cost of distillation.

Microorganisms can be used for aspects of all three processes. In thefirst case, microorganisms can assimilate the synthesis gases, such asCO₂, CO and H₂ into ethanol or acetic acid, or into longer chain alkylalcohols (e.g. 1-propanol, 1-butanol) or longer chain carboxylic acids(e.g. propionate, butyrate). In the second case, organisms are used toproduce enzymes for the degradation of plant fiber and for fermentationof sugars into ethanol. In the third case, microorganisms are used toboth digest plant biomass and convert it to alcohols. Finally, microbialcultures that can both digest biomass (case 3) and assimilate gases intoalcohols (case 1) can be used. In this case, the gases that are producedby organisms in the digestion of the biomass can be converted to ethanolor other alcohols.

For either the first or the third process, or a combination thereof, twodesired characteristics of the microorganisms used are: 1) ability toconvert a large portion of the substrate (e.g. gases or biomass) to thedesired products (e.g. alcohols or acids), and 2) ability to continueproducing the desired product even in the presence of highconcentrations of those products. Currently, microorganisms are notavailable for conversion of synthesis gases to high concentrations ofalkyl alcohols. The ability to tolerate high concentrations of products,and to still produce more of the product at high concentrations (about5% to about 6%, by volume), would make it possible to produce theproducts in a way in which it is cost effective to separate and utilizethe products.

SUMMARY

The disclosed invention is for a process to produce products offermentation wherein the fermentation is controlled by establishingconditions that make it thermodynamically favorable to produce desiredproducts over other products that might otherwise be produced. Further,the invention comprises microbial cultures that produce specific desiredproducts for use in the process, and the invention comprises a processto enrich and isolate microorganisms that produce desired products ofthe fermentation.

One use of the process is to convert synthesis gases (e.g. CO₂, CO andH₂) to lower alkyl alcohol or desired organic acids under conditionsthat make it thermodynamically feasible or thermodynamically favorableto produce the desired products.

Another aspect of this invention comprises selected microbial culturesthat can produce efficiently lower alkyl alcohols including ethanol,propanol or butanol from synthesis gases. The volumes of ethanol incultures reached at least, 1%, more preferably at least about 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 11% or 12%.

The volumes of butanol in culture were at least 0.5%, preferably atleast about 1%, 2%, 3%, 4%, 5%, 6% or 7%.

The volumes of propanol in culture were at least 0.5%, preferably atleast about 1%, 2%, 3%, 4%, 5%, 6% or 7%.

These microbial cultures convert a large portion of the gas mass to thedesired alkyl alcohol. For example, in one embodiment, up to at leastabout 95% of VFA plus lower alcohol content was ethanol plus butanol,especially 1-butanol.

The microbial cultures are also tolerant to the alkyl alcohol, andcontinue to grow in the presence of high concentrations of alkylalcohol, and continue to produce alkyl alcohol in the presence of highconcentrations of alkyl alcohol.

A further aspect of the application comprises microbial cultures thatcan both degrade biomass such as cellulosic biomass to alkyl alcohol,and can assimilate produced and perfused gases to produce additionalalkyl alcohol.

In addition, another aspect of the application invention comprises aprocess for producing cultures of microorganisms that convert a highpercentage of the biomass to a desired alkyl alcohol, and can toleratehigh concentrations of the desired alkyl alcohols.

Another aspect of the application comprises producing products fromsynthesis gases (CO₂, CO and H₂) using undefined mixed cultures, inwhich a mixture of products can be produced.

In addition to alcohols, co-products that can be produced with theprocess include: carboxylic acids such as volatile fatty acids (“VFA”),which can be converted to other products or used for various purposes,and microbial protein, which can be used as an animal feed.

This application also comprises a method for production of specific VFAor longer carboxylic acids, which can be separated and used for otherindustrial processes or converted to other products.

DESCRIPTION OF THE DRAWINGS

FIG. 1 indicates the change in free energy (ΔG, kJ/mol) for synthesis ofethanol, acetate or methane from H₂ and CO₂ as the molar ratio of H₂ toCO₂ increases. This figure shows the energy available for formingdifferent products peaks at a ratio of about 2 to 4. The model assumesthe process takes place at 1 atmospheric total pressure, 0.1 atmospheresmethane, and 0.001 M each aqueous acetate and ethanol, at 39° C., pH6.5.

FIG. 2 indicates the change in free energy (ΔG; kJ/mol) for synthesis ofalkyl alcohols from H₂ and CO₂ as the molar ratio of H₂ to CO₂increases. This figure shows that energy for forming alcohols isgreatest for longer alcohols at the ratio for maximal synthesis (3:1 forH₂ to CO₂), but otherwise shorter alcohols are favored over longeralcohols. Model assumed 1 atm. total pressure, 0.001M aqueous ethanol,1-propanol and 1-butanol, temperature 40° C., pH=6.5.

FIG. 3 indicates the change in free energy (ΔG; kJ/mol) for synthesis ofcarboxylic acids (C₂ to C₆) from H₂ and CO₂ as the molar ratio of H₂ toCO₂ increases. This figure shows the increase in energy available tomake longer carboxylic acids at the ratio for maximal synthesis (between2:1 to 3:1 for H₂ to CO₂), but that shorter carboxylic acids are favoredat both lower and higher ratios of H₂ to CO₂. Model assumed 1 atm totalpressure, 0.001M aqueous carboxylic acids, temperature 40° C., pH=6.5.

FIG. 4 indicates the calculated equilibrium concentration (mol/L) ofethanol or acetate at pH 4 where partial pressure of hydrogen (H₂)increases with constant total gas pressure of 1 atm comprising H₂ andcarbon dioxide (CO₂) gas. This figure shows that microorganisms wouldhave limited capacity to obtain energy from synthesizing ethanol fromsynthesis gases at 1 atm total pressure, and ethanol and acetatesynthesis are favored over degradation at specific ratios of H₂ to CO₂.

FIG. 5 indicates the calculated equilibrium concentration (mol/L) ofethanol or acetate at pH 4 where partial pressure of hydrogen (H₂)increases with constant total gas pressure of 2 atm comprising H₂ andcarbon dioxide (CO₂) gas. This figure shows that microorganisms wouldhave greater capacity to produce ethanol or acetic acid when underpressure especially when the ratio of H₂ to CO₂ is 2:1 or 3:1 foracetate or ethanol respectively.

FIG. 6 indicates the equilibrium concentration of ethanol or acetate atpH 4 where total pressure of all gases is increased and gases arecomprised of a constant ratio of 75% H₂ and 25% carbon dioxide (CO₂).This figure shows the greater feasibility to make higher concentrationsof ethanol at higher pressure.

FIG. 7 indicates the ratio of equilibrium concentrations of ethanol toacetate at pH 4 where total pressure of all gases is increased and gasesare comprised of a constant ratio of 75% H₂ and 25% carbon dioxide(CO₂). This figure shows the shift from the acid to alcohol productionwhen greater gas pressures are used.

FIG. 8 indicates the equilibrium concentrations (mol/L) of butyrate andbutanol at pH 4 where partial pressure of hydrogen (H₂) increases withconstant total gas pressure of 1 atm comprising H₂ and carbon dioxide(CO₂) gas. This figure shows the ratio for maximal butyrate or butanolsynthesis.

FIG. 9 indicates the equilibrium concentrations (mol/L) of butyrate andbutanol at pH 4 where partial pressure of hydrogen (H₂) increases withconstant total gas pressure of 2 atm comprising H₂ and carbon dioxide(CO₂) gas. This figure shows the dramatic effect on equilibrium ratiosof products as the pressure is increased.

FIG. 10 indicates the equilibrium concentrations (mol/L) of propionateand propanol at pH 4 where partial pressure of hydrogen (H₂) increaseswith constant total gas pressure of 1 atm comprising H₂ and carbondioxide (CO₂) gas. This figure shows the optimal ratio for propionateand propanol synthesis from synthesis gases.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Term Definitions

Aerotolerant: means the microorganism is able to grow in the presence ofopen air, such as an open flask because oxygen is not toxic to theorganism.

Alcohol tolerant: means that the microorganism is able to grow in thepresence of alcohols. Generally this means an amount of total alcohols(e.g. ethanol+propanol+butanol) of at least about 0.5% to about 1% byvolume, and preferably about 2% by volume, in an aqueous medium.

Butanol tolerant: means that the microorganism is able to grow in thepresence of butanol. Generally, this means an amount of butanol of atleast 0.5% to 1% by volume, and preferably about 2% by volume of aqueousmedium.

Carboxylic acid: means an organic compound containing the carboxyl groupCOOH or COO⁻ making it an organic acid because the proton (H⁺) can bedonated. Carboxylic acids range in length from 1 to many carbons, suchas greater than 20 carbons. Carboxylic acids are also called organicacids. The short-chain carboxylic acids (C₂ to C₅) are also calledvolatile fatty acids (VFA). Carboxylic acids are readily interconvertedwith their conjugate base (acid having released a proton to solution) inaqueous solutions and thus production of the acid or the base form isconsidered production of either form as they can be readilyinter-converted by adjusting pH of the solution.

Conjugate base: is one of two members of a pair of compounds that can beinterconverted by gain or loss of a proton (H⁺). The conjugate baseaccepts a proton from solution wherein the conjugate acid donates aproton. For example, for acetic acid the acid form is referred to as theconjugate acid and acetate is referred to as the conjugate base. Nearneutral pH (e.g. about 5 to about 7), most acid-base pairs of volatilefatty acids are predominantly in the conjugate base form. Furthermore,when free energy is calculated based on acid and base concentrations,the concentration of conjugate base was used with the associatedconcentration of protons (H⁺). A process that produces an acid or itsconjugate base and a proton are considered equivalent because the twoforms are readily interconverted.

Defined cultures: Cultures of microorganisms that have been isolated andat least partially characterized e.g. possibly identified as genus andspecies, or phylogenetically characterized by sequencing the variableregion of 16S rRNA, or by sequencing the complete genome.

Direct evolution: means to direct the development of microorganisms thatare well suited, preferably particularly well suited, for a givenenvironment that is different from the environment from which theorganism was taken, thereby changing the organism to be better suited tothe new environment.

Directed equilibrium: means a process in accordance to the invention inwhich a system is allowed to move toward equilibrium, but concentrationsof reactants and products within the system are manipulated, andpossibly some reactions are directly inhibited, to direct the system toproduce different products than would otherwise be produced asequilibrium is approached.

Ethanol tolerant: means that a microorganism is able to grow in thepresence of ethanol. Generally, this means an amount of ethanol of atleast about 0.5% to about 1% ethanol by volume, and preferably about 2%by volume of aqueous medium.

Favor: means the concentrations of reactants and products for competingreactions in the system, such as fermentation, are such that a greaterdecrease in free energy (more negative AG) results from one reactioncompared to another, where the first reaction is said to be favored overthe other or others. For example, synthesis of acetate may be said to befavored over synthesis of ethanol under certain conditions, oralternatively acetate synthesis may be said to be favored over acetatedegradation under certain conditions.

Favorable Free Energy for Synthesis: means the change in Gibbs FreeEnergy (ΔG) is negative for the combination of reactions that comprisethe system that converts a set of reactants to a set of products, andthe system can therefore convert the reactants to products. The ΔG iscalculated based on the change in Gibbs Free Energy under standardconditions)(ΔG° of temperature and the concentrations or partialpressures of reactants and products. The ΔG° is calculated as thedifference in Gibbs Free Energy of Formation)(ΔG°_(f)) for the productsand reactants. The ΔG°_(f) is the ΔG for formation of any material fromthe elements i.e. graphite, H₂, O₂, for example, under standardconditions. Standard conditions means standard temperature (298.15Kunless otherwise indicated), 1 molar concentration of all solutes ofreactants and products and 1 atmosphere partial pressure of gasescombined.

Fermentation or fermentation system: refers to the use of microorganismsto produce a product by, for example, the conversion of infused gases toacetate or ethanol; where the fermentation or the fermentation systemrefers to the totality of all possible reactions which occur duringdigestion.

Isolated microorganisms: means one or more microorganisms that eitherhave been isolated from a natural environment and grown in culture, orthat have been developed using the methodologies from the presentinvention and grown in culture. Some are highly pure, originally fromsingle, picked colonies. However, in the context, ‘isolate’ can refer toa culture enriched for a bacterium or bacteria with desired properties,where the desired bacteria is at least 5% of the culture, preferably10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

Lower alkyl alcohols: means C₂ to C₅ alcohols, i.e. ethanol, propanoland butanol.

Mixed cultures: More than one isolate of microorganism culturedtogether, may be defined or undefined, pure or impure cultures.

Molar proportion: means the molar concentration of one product as aproportion of the molar concentration of all of a type of product. Forexample, the molar proportion of butyrate of 50% of all volatile fattyacids means that the number of moles per liter of butyrate is 50% of thetotal moles per liter of all volatile fatty acids.

Molar ratio: Means the molar concentration of one product over the molarconcentration of another product. For example, a molar ratio of 1 forethanol to acetate means the concentration of ethanol in moles per literis equal to the concentration of acetate in moles per liter. The molarratio of gases can also be determined based on the moles of gas per unitvolume and pressure of the total gas.

Partial pressure of a gas: means the pressure a given species of gas.For example, if the total gas pressure is 1 atmosphere (atm) and carbondioxide comprises 20% of the total gas by volume, the partial pressureof carbon dioxide would be 0.2 atm or 20% of the total gas pressure.

Plant fiber: Defined chemically as comprising cellulose, hemicellulose,pectin or lignin, or combination thereof, and found in plant cell walland many forms of feedstock including whole plants, biofuel crops (e.g.switchgrass, algae), food byproducts, wood, wood byproducts, paper,waste, animal manure, human manure, and others.

Propanol tolerant: means that a microorganism is able to grow in thepresence of propanol. Generally, this means an amount of propanol of atleast 0.5 to 1% propanol by volume, and preferably about 2% propanol byvolume in aqueous media.

Pure cultures: Cultures of microorganisms that have been isolated orpartially isolated to eliminate contaminant microorganisms. Cultures canbe a single isolate or multiple isolates (mixed cultures).

Rumen microorganisms: means any or all of the microorganisms found inthe rumen of ruminant animals. This includes a diverse array of archaea,bacteria, protozoa, and fungi that digests fibrous plant material andferments starches and sugars, for example. Many of these organisms alsouse metabolites transferred from other organisms such as sugars releasedby digestion, VFA exported from other organisms, or H₂ and CO₂. Thisterm also includes such microorganisms that are also found elsewhere inaddition to the rumen including the digestive tract of animals, feces,silages, sludge, or in soil among other places.

Synthesis gases: means gases used to synthesize products. In the presentinvention the synthesis gases are usually carbon dioxide (CO₂), carbonmonoxide (CO), and hydrogen gas (H₂).

Thermodynamically favorable: means the concentrations of reactants andproducts are such that the reaction is favored over other reactions.

Thermodynamically feasible: means the process can proceed spontaneouslyin the forward direction according to the second law of thermodynamics.In a thermodynamically feasible reaction, the multiplicative product ofreaction product concentrations divided by the multiplicative product ofreactant concentrations is low enough for the reaction to proceedspontaneously in the forward direction according to the calculation ofthe ΔG for the reaction. Observation of a reaction proceeding in theforward direction indicates that the reaction is feasible inconsideration of all linked processes that enable the reaction to occur.

Total gas pressure: means the gas pressure in the fermentation systemincluding all gases whether added to the process or produced in thefermentation.

Undefined cultures: means cultures of microorganisms taken from a sourcewithout having isolated individual microbes or characterized individualorganisms.

VFA: means volatile fatty acids (e.g. acetic acid, propionic acid,butyric acid, iso-butyric acid, valeric acid, isovaleric acid, succinicacid, and lactic acid).

Basis of the Invention

This application is based in part on the use of the second law ofthermodynamics to control fermentation and on the discovery ofmicroorganisms that can convert CO₂, CO and H₂ to a much higherconcentration of ethanol or other alcohols than previously known. Forexample, the concentration of ethanol can exceed about 10% by volume orthe concentration of 1-butanol or 1-propanol can exceed about 6% byvolume. In addition, nearly all the synthesis gases can be converteddirectly to alcohol with little production of acetic acid or otherbyproducts.

Models of fermentation developed by the inventor form the basis of theprocess. These models incorporate the laws of thermodynamics andkinetics to explain and predict the profile of products fromfermentation. The models make it possible to establish conditions in afermentation to favor desired products and to select and improvemicroorganisms that make those products.

All chemical reactions are controlled kinetically or thermodynamically.With kinetic control, the rates of reactions depend on substrateconcentrations or enzyme activities, and these enzyme activities in turndepend on microbial growth or enzyme synthesis. The profile of productsformed depends on the relative rates of the different competingreactions. With thermodynamic control, the rates of reactions and whichpathway branches and direction are available depend on theconcentrations of reactants and products.

Biologists have focused on controlling kinetic elements of fermentationsuch as enzyme function, microbial activity, gene expression, orprovision of substrates. However, the inventors discovered thatfermentation is often controlled by thermodynamics. For example, in amixed-culture anaerobic digester, as soon as a glucose molecule isreleased by digestion of cellulose there are several microorganisms thatcan transport it into their cells and metabolize it to any number ofproducts. The amount of energy any particular organism can obtaindepends on the concentration of all the products of the reactionrelative to all the reactants. Since the free glucose concentration isvery low due to competition among microorganisms in the fermentation,and the products of fermentation are removed slowly, only very efficientmicrobes can use the small amount of glucose at all. And they can onlyuse it when concentrations of the products they produce are low.Therefore, when their products start to build up, they can no longerobtain energy by converting the reactant to a product, and they leavethe glucose behind for another microbe that produces a differentproduct. In this way, a constant ratio of products is produced.

In chemistry, whether or not a reaction can proceed spontaneously in theforward direction is represented by the change in free energy (ΔG),which can be calculated based on the ratio of products and reactants inthe system. Using this calculation, a strongly negative ΔG indicatesthat the reaction could proceed strongly in the forward directionwithout the addition of energy to the system. A strongly positive valueof ΔG indicates the reaction can not proceed in the forward directionwithout addition of energy to the system, and it may even run in thereverse direction.

If one calculates the ΔG values between many of the products in therumen of a cow assuming typical metabolite concentrations, one findsthat they are usually very near to 0. Additional products cannot be madeunless the reactant concentrations increase or the productconcentrations decrease. If one product increases, ΔG for that reactionincreases, thus different products of which ΔG is lower can be produced.The inventors developed mathematical models incorporating this knowledgeby solving multiple simultaneous equations using thermodynamic data topredict concentrations of products that would result. These modelsinclude competition for substrates and intermediates.

This work was undertaken to understand and mathematically modelfermentation in the cow's rumen. However, this basic research led toapplied research discoveries and inventions related to biofuelproduction. The inventors developed a process called “directedequilibrium”. Considering a system that can make many differentproducts, if these products are limited by thermodynamics, one can addundesired compounds to the media and make it thermodynamicallyinfeasible to produce more of those undesired compounds. Thus, only thedesired products will be made, and only organisms that make the desiredcompounds will survive. If it is not possible to find conditions tospecifically favor a certain desired metabolite, one can also use enzymeinhibitors to shut off undesired pathways that are difficult tomanipulate by thermodynamics. Thus, it is possible to control at leastsome of the products or reactants in the system to direct the system toa different equilibrium. In this way, the inventors have been able todiscover several useful microorganisms for different industrialprocesses.

Directed Equilibrium Process

The inventors calculated the change in free energy (ΔG) for manydifferent reactions in different fermentation systems and found thatafter accounting for some energy for ATP synthesis, the ΔG was near 0for the interconversion among many end products. The calculationsfurther showed that some reactions are near equilibrium even with verylow concentrations of some products. For example, ethanol was found tobe near equilibrium in rumen fermentation even though there is verylittle ethanol in the fermentation liquid. This observation showed thatthe reason for the low concentration of ethanol in the rumen was that itwas not a thermodynamically favored product. The inventor concluded thatchanging the concentrations of other products such as hydrogen or aceticacid would result in greater ethanol concentration. In fact, experimentsdemonstrated this point.

Once the reason for the low concentration was determined, and theconditions to favor ethanol production were identified, the conditionsof the fermentation could be altered to select for organisms thatproduce the desired alcohol. The isolated microorganisms could befurther developed by growing them under conditions in which ethanolproduction was thermodynamically favored over other products. Forexample, an organism found to produce both ethanol and acetate can begrown in H₂ or CO gas headspace, or with high acetic acid concentration,so that organisms that produce more ethanol and less acetic acid aremore fit. Over many generations, the culture selects itself into aculture that produces more ethanol from the gases presented, or aselective process is undertaken in accordance to the invention.

In general, the steps of the Directed Equilibrium process include any orall of the following:

-   -   1. Obtain a culture of microorganisms, which includes activity        that enables the conversion of a substrate to the desired        product.    -   2. Determine the association of all co-products for all end        products for and desired end products based on the stoichiometry        of balanced chemical reactions.    -   3. Calculate the ΔG for conversion of fermentation substrates to        each observed product and the desired product. To calculate the        ΔG, determine the ΔG° for formation of each product and reactant        from the elements. These values are typically found in        textbooks. The ΔG° (the free energy under standard conditions)        is determined for each reaction based on the stoichiometry of        each reaction for conversion of the substrate to each product        and the ΔG° of formation for products and reactants. The ΔG is        determined by adjusting the ΔG° to the temperature of the        fermentation, and using the actual final concentrations for each        product and reactant. Although this step can be enormously        helpful it may not be necessary to formally calculate        equilibrium concentrations or ΔG.    -   4. Determine alternative conditions that will shift fermentation        to thermodynamically favor production of the desired product.        These conditions may include inhibitors of pathways that are        otherwise favored, or addition of undesired products or gases or        removal of desired products or gases. This aspect of the process        can be aided by using a mathematical model in a spreadsheet        developed as part of the previous step. Alternatively, adding        inhibitors to reactions that produce competing products (e.g.        use same substrates) or adding concentrations of alternative        products will favor production of the desired product. The basis        of the response is the fact that the fermentation approaches        equilibrium, whether it is calculated or not, so other end        product concentrations prevent competing reactions.    -   5. Incubate the feedstock with microorganisms while maintaining        the conditions to shift the fermentation toward producing more        of the desired product. This step may require continuous        infusion or removal of metabolites or gases to make the desired        product thermodynamically favored over other products.    -   6. Whereas it may be cost prohibitive to continuously maintain        conditions to produce the desired product over all other        products, microorganisms that produce the desired product will        be enriched for over time decreasing future competition for        other products. These microorganisms grow faster under the        conditions that favor production of the products they make, so        diluting all organisms repeatedly overtime results in        disappearance of organisms that produce undesired products.    -   7. An alternative approach to the previous step is to use        conditions that favor production of the desired product but        which may not result in its accumulation. For example, including        an ethanol-degrader in the fermentation and conditions leading        to ethanol production and subsequent degradation (e.g. high H₂,        low CO₂) would manage to keep ethanol producers in the culture,        and they could be further enriched or cultured in a subsequent        step.    -   8. To select a pure culture of microorganisms to produce mainly        the desired product, the enriched culture is diluted serially        and plated or a roll tube is produced to grow them in an agar as        individual colonies. Thermodynamic conditions (e.g.        concentrations and partial pressures of gases) are used so that        only the desired organisms can grow. Colonies that grow are        selected, purified and tested to use them under conditions        wherein they can produce the desired product based on the        thermodynamic model.    -   9. Even a pure culture of a microorganism might produce a wider        array of products than desired, or may not produce a high        concentration of the desired products. Directed Evolution can be        conducted by subjecting the pure culture to fermentation on the        feedstock to be digested or substitute feedstock while        controlling the products and reactants to make it        thermodynamically favorable to produce the desired product over        other products. Over many successive generations, as described        previously, mutant organisms thrive and other organisms are        diluted or washed out.    -   10. Another aspect of the process includes growing the        microorganism with a high concentration of the desired product        to select for organisms that can tolerate such a high        concentration. Furthermore, conditions can be used to make it        thermodynamically favorable to degrade the desired product. Such        organisms might be isolated while degrading a compound, and        later be grown under different conditions wherein producing the        product is thermodynamically favored and under those opposite        conditions, the product may be produced.    -   11. Furthermore, the microorganisms selected can be made more        tolerant to the desired product by growing them in the presence        of increasing concentrations of the desired product while        maintaining conditions to make it favorable to continue        producing the desired product in the presence of the high        concentration.    -   12. Another aspect of the invention is to use the second law of        thermodynamics to analyze the fermentation system, including an        industrial fermentation and each of the organisms in it, to        understand what combinations of organisms can digest feeds to        certain concentrations of products, and thus understand how to        use the optimal organisms for all components of the feedstock        available for the products that are desired.

The directed equilibrium process when used to enrich and isolatemicroorganisms differs from previously known processes in that amicrobial system is analyzed using multiple simultaneous equations basedon the second law of thermodynamics to develop conditions wherein onlyorganisms that produce a certain product can survive or, at least aremore fit than undesired organisms. Previously known systems forisolating microorganisms have used the starting mixed culture andcertain substrates to enrich or isolate organisms that could use thosesubstrates, but a wide array of products could result. Often, noorganisms that produce the desired product were isolated because theconditions (pH, temperature, gas composition, metabolite accumulation)selects against the desired organisms.

The present invention applies a newly discovered principle, which is notyet widely understood or accepted, that microbial ecosystems approachthermodynamic equilibrium. The inventors discovered this principle andapplied it to control microbial ecosystems and enrich and select formicroorganisms that produce a desired product. Using mathematical modelsemploying the laws of thermodynamics, it is possible to select anddevelop microorganisms for many different processes.

Production of Alcohols and Acids from H₂, CO₂, and CO

One embodiment of the current application is the production of loweralkyl alcohols from H₂, CO₂ and CO using the directed equilibriumprocess. Using a ratio of H₂ to CO₂ or H₂ to CO or both determined bythermodynamic analysis to make greater concentration of the desiredproduct possible, and to favor the desired product over undesiredproduct, drives the reaction toward the desired alcohol or desired acid.In this way, a higher concentration of the desired product, and a lowerconcentration of the undesired product, is obtained and a greaterpercentage of the gas is converted to the desired product.

In addition, using elevated pressures of the gases, including totalpressure greater than 1 atm and preferably greater than 2 or even morepreferably greater than 4 atm, makes the synthesized products (e.g.ethanol) thermodynamic ally more favored than degradation of theproducts back to gases. Increasing pressures also shifts fermentationtoward alcohols over acid production, and toward longer chain-length ofacids (e.g. butyrate or valerate) and longer chain-length of alcohols(e.g. butanol).

The pH in the process is controlled to optimize microbial growth andconversion efficiency to alcohols. For example, pH 5 favors alcoholproduction over acid production more than pH 7 with other conditionsbeing equal, but at pH 5 microbial growth may be decreased. Depending onthe conditions and organisms, alcohol production may best be achieved atpH from about 4 to about 7, or even lower than 4.

The inventors were able to isolate microorganisms that could synthesizealcohols and volatile fatty acids from CO₂, CO and H₂. In the process,the concentrations of infused gases and other products are adjusted toratios that make production of the desired products thermodynamicallyfavorable, and partial pressures are increased, so that highconcentrations by volume of the desired product can be produced. Forexample, ethanol was produced in media with greater than 10% ethanolconcentration by volume.

Second, the adjustment in gases and other metabolites increases thepercentage of the infused gases that is converted to the desiredproduct. A higher partial pressure of H₂ relative to CO₂ or CO favorsgreater conversion of gases to alcohols rather than acetic acid. Forexample, under certain conditions ethanol production isthermodynamically feasible while acetate production is not, and ethanolis formed with little production of acetate.

Third, microorganisms that are used for this process, isolated as anaspect of the present invention, were found to increase the percentageof gases converted to the desired product compared to existingmicroorganisms, and these isolated microorganisms have greater toleranceto the desired product. Some isolated microorganisms produced ethanolfrom CO₂ released from biomass digestion and added H₂ to greater than 7%ethanol by volume. Other isolated organisms produced ethanol, 1-propanoland/or 1-butanol from added CO₂ and H₂ in media with 10% ethanol, andwere tolerant and grew in media with as much as 10% ethanol, 6%1-propanol, or 6% 1-butanol.

Fourth, these improvements and the isolated microorganisms also increasethe rate (i.e. unit product per liter per unit time) in which thedesired products can be produced.

Fifth, in addition to production of acetate and ethanol, longer chainalcohols and acids can also be produced from the synthesis gases. Forexample, some isolated organisms and processes predominantly producedbutyrate rather than acetate. Some isolated microorganisms producedsignificant amounts of iso-butyrate and iso-valerate from CO₂ and H₂ orCO and H₂. Rumen microorganisms are known to make longer-chaincarboxylic acids including odd-chain length carboxylic acids. Theinventors are making caproic acid (C₆), caprylic acid (C₈), capric acid(C₁₀) or longer carboxylic acids as well.

Sixth, microorganisms may be improved through a process of directedevolution described as an aspect of the present invention. Theseimprovements pertain to producing more of a desired product as afraction of all products, at a higher concentration, and at a higherrate per unit time.

Another aspect of the invention includes using mixed cultures ofmicroorganisms that can produce one or many different products. Thesemay be undefined cultures or mixtures of pure cultures.

Some industrial processes produce low concentrations of the synthesisgases among other gases like nitrogen or air. By adjusting the ratio ofCO₂, CO and H₂, or increasing total gas pressures, these relatively lowconcentrations can be used more effectively for synthesis of desiredcompounds. In fact, simply increasing the total pressure makes itpossible to produce products from residual gases from industrial oragricultural processes that would otherwise be discarded. For example,1% H₂ could be used in this process more easily than it may be recoveredfor other use.

These improvements to methods to make alkyl alcohols from gases,adjusting ratios of gases and increasing pressure, increase alcohol oracid concentrations when using cultures of microorganisms that havealready been isolated and may or may not already be considered forindustrial production of acids or alcohols. These improvements alsoincrease the portion of gas converted to the desired substrate.

Using microorganisms isolated from the rumen of a cow, but which couldbe isolated from many other environments, allows for production of alkylalcohol at greater concentration of ethanol or other alkyl alcohol thanpreviously disclosed. The microorganisms already isolated, and that canbe isolated, are an aspect of this invention. Methods to isolate themicroorganisms that can be used for this process are another aspect ofthe invention.

Process to Calculate Free Energy and Equilibrium Concentrations

A mathematical model defined in a spreadsheet is used to determine thechange in free energy for different reactions that may occur infermentation. The model may be modified by adding or subtractingreactions as warranted by different types of fermentation. In addition,the free energy change for reactions can be determined for differentconditions (e.g. temperature, pressure, pH, concentrations ofmetabolites, pressures of gases). In addition, the equilibriumconcentrations or equilibrium ratios of metabolites can also bedetermined. The description that follows provides the informationnecessary to create the model, or a similar model for differentmetabolites that can be included.

The balanced reactions giving rise to each potential product from thebiomass source are first determined. For example, acids, alcohols andalkanes like methane can be derived ultimately from CO₂ and H₂. Thestoichiometry is determined by balancing each reaction so that equalnumbers of carbon, hydrogen, oxygen and so forth are on each side of theequation. For example,

CO₂+4H₂←→CH₄+2H₂O

2CO₂+4H₂←→CH₃COOH(a.k.a. acetate)+2H₂O

3CO₂+7H₂←→CH₃CH₂COOH(a.k.a. propionate)+4H₂O

4CO₂+10H₂←→CH₃CH₂CH₂COOH(a.k.a. butyrate)+6H₂O

2CO₂+6H₂←→CH₃CH₂OH(a.k.a. ethanol)+3H₂O

3CO₂+9H₂←→CH₃CH₂CH₂OH(a.k.a. 1-propanol)+5H₂O

4CO₂+12H₂←→CH₃CH₂CH₂CH₂OH(a.k.a. 1-butanol)+7H₂O

CH₃COOH(a.k.a. acetate)+CO₂+3H₂←→CH₃CH₂COOH(a.k.a. propionate)+2H₂O

2CH₃COOH(a.k.a. acetate)+2H₂←→CH₃CH₂CH₂COOH(a.k.a. butyrate)+2H₂O

CH₃COOH(a.k.a. acetate)+CH₃CH₂COOH(a.k.a. propionate)+2H₂←→

CH₃CH₂CH₂CH₂COOH(a.k.a. valerate)+2H₂O

Thus, the balanced equations can be determined even without knowledge ofthe exact pathway. The respective pathways are determined for any andevery reaction thought to occur in the fermentation system of interest.Which reactions occur can be assumed based on what products accumulateor are otherwise found in the fermentation.

The ΔG for any pathway depends on which products and reactants areproduced, and therefore different conditions are needed to make eachreaction thermodynamically feasible. The change in Free Energy understandard conditions)(ΔG°) is determined in the established way ofcalculating the Free Energy of Formation from the basic elements foreach reactant and product and subtracting the Free Energy of Formationof the products from the Free Energy of Formation of the reactants(Chang, R. 1981. Physical Chemistry with Applications to BiologicalSystems: Second Edition, MacMillan Publishing Co., Inc., New York, whichis incorporated herein by reference). For the current patentapplication, the free energy of formation values not found in the bookauthored by Chang were obtained from the literature (Guthrie, J. Peter;1992. A group equivalents scheme for free energies of formation oforganic compounds in aqueous solution. Canadian J. Chemistry70:1042-1054 which is incorporated herein by reference). The relevantvalues from the literature are provided again in Table 1. Similarinformation can be obtained from these references and others if desiredto add other metabolites to the model.

The values in Table 1 are the relevant thermodynamic data under standardconditions for these reactants and products as well as some otherimportant potential fermentation intermediates. These values representthe free energy of formation)(ΔG°_(f)) and enthalpy of formation(ΔH°_(f)) of the metabolites from the elements (e.g. H₂, O₂, graphite).Free energy)(ΔG°) and enthalpy (ΔH°) under standard conditions andconcentrations can be determined from these tabular values for eachreaction of interest (Chang, 1981 as cited). Standard conditions are 1 Mconcentration of each soluble reactant and product, 1.01325×10⁵ Pa (1atm) of all gases, and 298.15K.

ΔG°=ΔG° _(f) of products−ΔG° _(f) of reactants, and

ΔH°=ΔH° _(f) of products−ΔH° _(f) of reactants

Adjustment to each ΔG° for temperature can be made using atransformation of the van't Hoff equation and enthalpy where T₁ and T₂are the initial and final temperatures respectively, and ΔG°_(T1) andΔG°_(T2) are the respective standard free energy values:

ΔG° _(T2) =T ₂ /T ₁ [ΔG° _(T1) −ΔH°(T ₂ −T ₁)/T ₂]

So, for example the ΔG at 39° C. or 312K was determined for many of thereactions of interest from the tabular data reported at 298.15K becausethe fermentations were conducted at 312K.

Once the ΔG° is determined, it can be used to calculate the actual ΔGfor a specific set of conditions using the equation:

ΔG=ΔG°±RT In {[products]/[reactants]}

where the [products] and [reactants] are concentration of all productsor reactants in the fermentation, T is temperature in degrees Kelvin.For the current studies temperature was usually set to 312K. R is thegas constant=0.00831 kJ/K. Given the value of ΔG, the free energyavailable for a reaction can be shown. If the ΔG is negative, therewould be energy for organisms to produce ATP and grow while carrying outthe process. If the ΔG is positive, the opposite reaction might enableorganisms to obtain energy. Generally, about 44 kJ/mol is required forfermentation organisms to produce a mole of ATP, but many organisms andreactions can produce a fraction of an ATP and the exact requirement forfree energy depends on energy status of the organisms and other factors.

TABLE 1 Thermodynamic data of selected compounds. Substance ΔH°_(f)ΔG°_(f) Methane (g) −74 −50 Ethane (g) −84 −32 Methanol (aq) −201 −176Ethanol (aq) −235 −182 1-Propanol (aq) −255 −255 2-Propanol (aq) −273−186 1-Butanol (aq) −275 −163 2-Methyl-1-propanol (aq) −284 −1672-Butanol (aq) −293 −179 1-Pentanol (aq) −293 −179 Acetoaldehyde (aq)−294 −153 Acetic acid (aq) −432 −394 Propionic acid (aq) −453 −385Butyric acid (aq) −475 −378 Valeric acid (aq) −491 −365 Caproic acid(aq) −511 −386 Glucose (aq) −1264 −917 CO₂ (aq) −413 −386 H₂ (g) 0 0Water (l) −286 −237Free energy in kJ per mole under standard conditions of severalpotential fermentation metabolites at 298.15K and 1.01325×10⁵ Pa (1atmosphere). Standard conditions are 1M concentration of each solublereactant and product, 1.01325×10⁵ Pa (1 atm) of all gases, and 298.15K.

The inventors have taken the thermodynamics of fermentation to a wholenew level. They discovered that using actual concentrations of reactantsand products, many of the metabolites in fermentation are nearequilibrium with each other, or ΔG values are close to 0 for thepresumed interconversion of the end products. The explanation is that asone metabolite builds up, there is less energy that can be obtained bymaking more of that metabolite. Less energy means less ATP or microbialgrowth supported. Therefore, less of that product is made. Thisknowledge enabled the inventors to determine conditions that arenecessary to select for previously unknown microorganisms carrying outpreviously unknown pathways such as synthesis of medium-lengthcarboxylic acids and alcohols from CO₂ and H₂.

All of the separate equations for ΔG are compared for a given set ofconditions, and the conditions altered in a spreadsheet to determine theimpact on the ΔG for each reaction. In this way, conditions can bedetermined in which a desired reaction is favored (ΔG more negative)compared to other reactions.

In addition, the equilibrium concentrations of products are determinedassuming certain reactions might go to equilibrium and use availablesubstrate. For example, in a mixed culture fermentation includingmethanogens, with H₂ pressure set high and CO₂ pressure set low,degradation of VFA to CH₄ and CO₂ would be favored over VFA productionfrom CO₂ and H₂. Using the thermodynamic model requires theunderstanding that selection for conditions to accumulate a favoredproduct are not necessarily the same as conditions to enrich for orselect organisms that make that product. It may be advantageous toestablish conditions in which the favored product is created before itis converted to something else by different organisms. As long asconditions favor production of the desired product in the first place,they may be ideal for enrichment or isolation.

The equations are entered into a spreadsheet to solve simultaneously forseveral different potential reactions. In addition, several differentsets of conditions can be compared to determine what conditions arenecessary to shift fermentation from one metabolite to another. Forexample, many anaerobic microbial fermentations produce methane from CO₂and H₂. By altering the ratio of gases in the digester, one can showratios and pressures that favor production of acetate relative tomethane. The model may show that conditions to inhibit methane may benecessary in a mixed culture, or to show the ratio of gases that favormaximal acetate production.

When a reaction proceeds to equilibrium, the ΔG tends toward 0. The ΔGfor the simultaneous equations can be set to 0 or some negative valuerepresenting a certain amount of captured ATP (e.g. −44 kJ/mol of ATPgenerated) or a certain efficiency of a process. The equilibrium modelshows whether or not production of a certain product is feasible under acertain set of conditions. For example, the conditions necessary toproduce a certain concentration of ethanol from CO₂ and H₂ can becalculated. It is especially advantageous to use the integrated model todetermine the potential to produce a desired concentration of ethanol inthe system in which the soluble CO₂ and H₂ may be limited by thecompeting pathway to produce acetate from the gases. The equilibriummodel integrating multiple reactions can show the conditions necessaryto shift fermentation from acetate to ethanol. The simplest way tocalculate these conditions is to determine the ΔG for astoichiometrically balanced conversion of acetate to ethanol, and thenset the ΔG to 0 and solve for the ratio of ethanol:acetate underdifferent conditions. Using the equilibrium model, it is possible toshow a set of conditions wherein the acetate cannot be furtherconcentrated but the ethanol can be concentrated above 10% by volume ofthe media. Thus, the integrated model establishes conditions to isolateorganisms or to use isolated organisms to produce a high concentrationof ethanol or to shift from acetate to ethanol production.

The free energy of individual reactions is determined in chemistry andis occasionally used for industrial fermentation reactions. However, theinventors use these calculations in a far more sophisticated wayintegrating multiple reactions to determine conditions to shiftfermentation from one product to another.

Methods of Microbial Enrichment and Isolation

Once having established the conditions that make production of a certainproduct thermodynamically feasible or thermodynamically favorablecompared to other possible reactions, conditions are established tofavor production of certain products. These conditions are maintainedfor production, and they are used to enrich for certain microorganismsthat produce the desired products, and to isolate the organisms thatproduce the desired product.

The organisms selected and developed are robust (easily maintained andgrow quickly), produce the desired products (e.g. alcohols and acids) ata high rate to a high concentration of product, and convert a highpercentage of the substrate (e.g. gases) to the desired products. Ingeneral, isolation methods employ mathematical models incorporatingthermodynamics and kinetics to create conditions in which only thedesired organisms can grow.

Standard Microbiological Procedures

Generally, microbial selection is preceded with enrichment of desiredtraits. Organisms that have desired functional traits are enriched underspecific conditions for several periods of growth followed bysub-culturing and dilution. For example, conditions can be created thatfavor long-chain acid production so that only the long-chain acidproducers grow quickly. Selective media are inoculated with a mixedculture and incubated under those conditions for one to five days. Evenlonger lengths of time can be used to select slower growing organisms. Aculture starting with 10⁸ viable cells per ml may end with 10¹⁰ cellsper ml, and 99% of the new cells will have grown recently under therestrictive conditions. Therefore if a rare organism in the originalculture thrives under highly restrictive conditions, it will dominatethe new culture. For example, one in a million organisms, or 100 in ahundred million, could make up 99% of the culture after one enrichmentphase if only those organisms can grow. If the conditions only favordesired organisms, but do not completely exclude competitors, it takesseveral iterations of culturing and sub-culturing to reach a steadystate in which more organisms with the desired traits exist in theculture compared with the initial conditions. The inventors typicallyuse a few to more than 10 separate enrichment steps.

Often different conditions are used in alternating enrichment steps. Oneset of conditions may be used to select for a certain functional trait,and a second set of conditions used sequentially for a second functionaltrait. For example, if we desire organisms that produce alcohols we mayuse one set of conditions to select for alcohol production from gasesand a second set of conditions for production of alcohols fromcellulosic biomass. The result will be organisms that can producealcohols from both gases and cellulosic biomass

Once the desired traits have been enriched, individual isolates of theorganisms are selected. Serial dilutions from 1 to 10⁻¹⁴ are poured toagar plates, which are incubated under restrictive conditions. Theinventors incubate agar plates with specific gas compositions andpressures. The inventors also use roll tubes at times to apply certaintypes of gas composition.

For roll tubes, warm agar is inoculated in test tubes, which are sealedand rolled on ice to make the agar gel. The agar hardens around theperimeter of the tube. Both agar plates and roll tubes typically areincubated at 40° C. until discernable colonies form, usually within oneto three days. Higher or lower temperatures can also be used to obtainorganisms that thrive at different temperatures.

Colonies are picked from the tubes and plates using sterile technique.The plates and tubes with less diluted inoculation have overlappingcolonies, while the ones that are highly diluted do not have anycolonies. Some plates have individual colonies, and these are added to abroth and incubated under conditions to favor production of the desiredproduct. The inventors used 32-liter canning pressure cookers asanaerobic chambers. Several racks of test tubes or agar plates can beadded to the chambers with adequate headspace so that gases do not needto be changed more than once a day. The chambers support up to 3atmospheres of gas infused through a valve into the chamber. Inaddition, the leading organisms were incubated in tubes or flasks withgases bubbled into each fermentation using small aquarium pumps insidethe chamber.

The products each microorganism isolate produces may be screened usingrapid procedures (e.g. pH after titration to a specific point withsodium hydroxide to determine total acid produced, or optical density toestimate microbial growth), and leading candidates are analyzed forseveral products using gas chromatography (GC) or other technique. Afteran initial screening of all isolates, the most promising isolates arefurther evaluated by determining the optimal conditions for their growth(e.g. pH, temperature, substrates, oxygen), and the rate of growth (g/Lper day), sensitivity to end products (e.g. acids, alcohols).

Following this process typically isolates and evaluates a few hundred toa few thousand organisms each run. The results are evaluated todetermine which end products and metabolic pathways dominate among thefavored isolates and potential weaknesses of the isolated microorganismsfor use in an industrial process. Once the best cultures are selectedand evaluated under many different conditions, the growth rate,production rate, conversion efficiencies (percent of CO₂ and H₂conversion to different products), titer and tolerance to potential endproducts are determined.

After evaluation of the leading isolates and identification of theweaknesses of each leading isolate, a process of mutagenesis can beinitiated. In this process, the conditions in which the desired productsare strongly favored are used on separate pure cultures of large numbersof organisms (e.g. 10¹⁰) of an isolate. Conditions are established inwhich production of desired products are favored and production ofundesired products are strongly disfavored. For example, if an organismonly produces a few products, the inventors can focus on the conditionsto select against those few undesired products. A range of conditionscan be used to both favor desired organisms and disfavor undesired ones.No organisms grow under many sets of conditions, while under otherconditions a few grow and they take over the culture. The survivors canthen be enriched again under even more restrictive conditions. In thisway, the mutants that have special abilities within the originally pureculture can survive. To increase the genetic diversity, some culturesare exposed to different levels of ultraviolet light briefly at thebeginning of the incubation. The treatment increases mutation rate andaccelerates the evolutionary process. The developed organisms areselected on agar plates and tested as previously. Thus, the entireprocess of enrichment and selection can repeated if necessary many timesfor both original selection and development.

Much of the emphasis in recent years has been on specific geneticmanipulation and transformation to engineer organisms. However,isolation and non-specific mutation methods are also importantespecially in the early stage of microbial development. First, thegenotype of the newly isolated organisms is not known, and in many casesplasmids or other vectors may not have been identified. Second, thetypes of changes that are desired may not be understood metabolically.For example, isolating organisms that are tolerant to highconcentrations of the desired products (e.g. specific acids), and growquickly or at least are robust are complex problems. These traits arecomplex and may require several genetic changes, many of which are notfully understood. Therefore, it would not be easy to begin specificmanipulations of DNA to create the desired organism. Furthermore, theinventors discovered it is often not necessary as there are manyorganisms that already evolved to thrive under the conditions needed inan industrial process. Therefore the inventors initially focus onenrichment, isolation and nonspecific mutation for obtaining organismswith the traits that are difficult to engineer, and then apply specificengineering to simpler DNA modifications later if necessary. Forexample, the inventors selected organisms that convert a high percentageof the gases to the desired product, and isolated organisms that producediffering amounts of desired products under certain conditions; theorganisms can obtain energy from producing the desired products. Ifnecessary, some of these organisms can be further manipulated throughspecific genetic engineering to knock out the ability to produceundesired products later. Once organisms are transformed, mathematicalmodels can again be used to select for the successful individuals and toestablish conditions that maximize the production of the desiredproducts from reactants.

Use of Mathematical Models

The inventors developed transformational improvements to microbiologicalmethods based on mathematical models of fermentation. The inventors useintegrated models that simultaneously use both thermodynamics andkinetics, and that solve simultaneous equations representing multiplepathways. For example, the approach predicts the consequences ofcompetition for substrates, and determines conditions in which organismsthat obtain energy from a certain pathway are or are not be able togrow. The mathematical models can be quite complex and all possibleresults cannot be presented here. However, all the information needed torecreate the models for production of any fermentation product ispresented in this application, and general conclusions from the modelswill be described below to illustrate the process for production ofalcohols and carboxylic acids from H₂ and CO₂.

One illustration relates to the Gibbs free energy change (ΔG) fordifferent reactions that can produce products from CO₂ and H₂. If the ΔGis negative, the reaction can proceed in the forward directionspontaneously. The most negative ΔG represents the reaction with themost favorable conditions to carry out the reaction. Reactions withpositive ΔG can proceed in the forward direction, but only if energy isput into the system to drive the reaction forward. In other words,another linked reaction with an even greater absolute value of ΔG thatis negative must be linked to make the overall ΔG negative. For endproducts relative to reactants like CO₂ and H₂, a positive ΔG means theorganisms that carry out the reaction will have to use their own energyto make the reaction proceed. If an organism is using energy to make thedesired product, it will not be able to grow from carrying out thereaction. On the other hand, an organism that carries out a reactionunder conditions with very strongly negative ΔG will not only be able tocarry out the reaction without using its energy stores, it will also beable to link ATP generation or other storage of energy. Thus, thestrongly negative ΔG will allow for the fastest growth rates(particularly with some types of organisms because the energy can begenerated from dissipation of proton gradients in continuous fractionsof ATP).

The inventors used this model to enrich and isolate organisms thatproduced carboxylic acids or alcohols in a high concentration and didnot produce as much acetic acid when it was not desired. The calculatedΔG depends on the ratio of products to reactants, and the inventorscalculated the ΔG for several different potential pathways. There arechanges to the fermentation that strongly change which products arefavored from H₂ and CO₂. One change is the ratio of H₂ to CO₂, andanother change is the combined pressure of both gases. The ratio of H₂to CO₂ that produces the highest possible concentration of acetic acidis 2:1, but the ratio to produce the highest concentration of alcoholsis 3:1. Carboxylic acids with greater than two carbons are produced inthe highest potential concentration with ratios of H₂ to CO₂ between 2:1and 3:1 with the longer acids favoring the higher ratio. The optimalratio to convert acetate to propionate is 3:1.

Increasing the total pressure of H₂ and CO₂, especially at the optimalratio, exponentially favors alcohols and long-chain acids over acetate.For example, at twice the pressure (2 atm vs. 1 atm) the equilibriumconcentration of acetate increases 70 fold, but the equilibriumconcentration of ethanol increases more than 250 fold. Thus, incubationunder moderate pressures of optimal ratios of H₂ and CO₂ shiftsequilibrium toward desired products and in fact, the organisms thatproduce those products will be favored. High ratio of H₂ to CO₂ alsofavors longer-chain carboxylic acids over acetate. For example, at twicethe pressure of total gases, the concentration of butyrate increases17,000 fold (compared to 70 fold for acetate), and the equilibriumconcentration of butanol increases 80,000 fold. The large numbers andhuge swings in directionality of reactions make it necessary to performthe calculations in order to define the conditions for synthesis of thedesired compounds. It is not surprising that previous investigators mayhave occasionally observed butyrate or butanol in fermentations withoutbeing able to repeat or confirm the observations. The conditions makingit thermodynamically favorable to make these products must be understoodin order to repeatedly and reliably produce the products. Theseprocedures have been tested and used already and the empirical resultsconfirmed the theoretical expectations.

In addition to these procedures to favor organisms that produce thesedesired products, the metabolic models the inventors are using point toseveral other ways to further select desired organisms. One surprisingapproach to obtain organisms that carry out a desired conversionreaction (e.g. A→B) is to isolate organisms that carry out the reverseof the desired pathway (e.g. B→A). In other words, one can enrich themunder conditions that start with a high concentration of the desiredproduct and favor degradation of the product. In a subsequent enrichmentphase, the desired product is removed and the thermodynamic conditionsare reversed. The resulting organisms can create the desired productunder one set of conditions and degrade it under another set. Thisapproach applied to enrichment of organisms selects for a high degree oftolerance to the desired product (only organisms that grow in a highconcentration survive) and a high specificity of production (selectedorganisms have the enzymes to make or degrade the desired product). Itis based on the theory that all catalysts decrease activation energy ofreactions, and they do not change the equilibrium constant or ΔG.Catalysts such as enzymes accelerate the rate of reactions when thosereactions are kinetically controlled, but they must also accelerate therate of the reverse reactions to an equal proportion. Otherwise theequilibrium constant would change. A corollary to this principle is thatall catalyzed reactions are bi-directional. If we seek enzymes tocatalyze a given reaction, for example to produce butanol, organismsthat degrade butanol have those enzymes. The cell machinery may not beset up to allow the organisms to grow (produce ATP) under both sets ofconditions, but many organisms can obtain energy by metabolizing thereaction in either direction.

A further application of the model to establish conditions forenrichment pertains to using aerobic conditions in the enrichment phase.Many organisms can aerobically catabolize a substrate like glucose toCO₂ and H₂O. However, they may also survive under anaerobic conditionsby making another end product that does not require oxidation. Forexample, they may make acetate and H₂ or ethanol or lactate. If oxygenis returned to the environment, they may further oxidize these “endproducts” to CO₂ and H₂O for additional energy. Thus, it is advantageousto isolate such facultative aerobes by growing them in the presence ofoxygen and high concentrations of the desired product. For example, theinventors used this technique for isolation of facultative aerobes thatproduced alcohols from H₂ and CO₂. In one phase of the enrichment, thealcohol was oxidized to CO₂ and H₂O, and in the other phase, organismswere grown under conditions to favor production of the desired acid fromH₂ and CO₂. There are special advantages of facultative aerobes. Theycan be grown aerobically very quickly while only producing CO₂ and H₂O,which can be easily removed. Even facultative anaerobes are advantageousbecause of their ease of handing in the laboratory and industrialprocess.

Methods of Producing Organic Products

Whether using an isolated pure culture of one or more microorganisms orusing an undefined mixed culture, the directed equilibrium processallows for production of a desired product in a higher yield relative toother products, at a higher concentration before showing signs ofapparent intolerance to end product, and at a consistently high rate. Inthis process, the thermodynamics of all possible pathways in thefermentation are determined and the conditions are established to favorthe desired pathways and produce the desired product. This process goesbeyond the calculation of the thermodynamic feasibility of producing onedesired product. All of the products, desired and undesired are includedin the calculations, and the ΔG for the interconversion of all endproducts is also calculated even if pathways for interconversion are notknown. These ΔG values estimate the feasibility of one reaction relativeto a different one, and if more than one product exists, conditions needto be determined to favor the desired product over the undesired one.Conditions are tested to determine the optimal conditions to favor thedesired pathway. One calculates whether the desired pathway is feasibleor not and whether undesired pathways are infeasible or disfavored. Inthis way, the optimal conditions are established and used for productionof the desired product.

A further aspect of the directed equilibrium process is that thedirection of pathways can be reversed by controlling the thermodynamicconditions. For example, a process to produce CO₂ and H, from acetate orlonger chain acids can be reversed to produce acetate from CO₂ and H₂.In many cases the same microbial culture can be used for bothdirections, but in other cases the conditions to isolate microbes needto be undertaken separately to optimally control the reaction in theopposite direction.

Directed Equilibrium Process to Produce Lower Alcohols and CarboxylicAcids from H₂, CO₂, and/or CO Microorganisms

One aspect of the present invention is a microbial culture that cantolerate high concentrations of ethanol, such as greater than 2% ethanolby volume, and more preferably greater than 6% ethanol by volume, andeven more preferably greater than 10% ethanol concentration by volume.The isolated organisms grow in the presence of these high ethanolconcentrations, and convert a high percentage of the gases CO₂, CO andH₂ to ethanol. The same organisms also tolerate high concentrations ofvolatile fatty acids, such as 3% total volatile fatty acids by volume orpreferably 4% or more preferably 5% VFA by volume. The organisms arealso tolerant to low pH, such as less than pH 5 and preferably less thanpH 4. However, the organisms generally also grow at neutral pH such aspH 7 or higher. In addition, some of the isolated organisms are tolerantto 6% 1-propanol by volume or 6% 1-butanol by volume, and they makethese alcohols when these concentrations of alcohols are present.

In addition, some isolated organisms can produce other products besidesalcohols from synthesis gases. These products include acetate andlonger-chain acids like propionate, and butyrate. The organisms toleratehigh concentrations of the end products. Certain isolates specialize inproducing alcohols like ethanol, while others primarily produce acertain VFA like butyrate from the synthesis gases. It is notnecessarily advantageous to isolate or develop microorganisms thatproduce many different products. If many different products areproduced, certain products may inhibit further fermentation, and it ismore difficult to separate several products. Thus, one advantage of theisolated microorganisms that comprise an aspect of this application isthat many isolated isolates specialize in producing few products. Forexample, they mostly produce alcohols or only short-chain volatile fattyacids (VFA) or mostly longer chain carboxylic acids. The inventorscontemplate methods for isolating organisms that produce any particularcarboxylic acid or alcohol for use in a specific process.

The present invention pertains to microorganisms that produce highconcentrations of alcohols or desired carboxylic acids from gases, acombination of CO₂ and H₂ or CO and H₂ or a combination of all three.Most organisms that were isolated to use CO₂ and H₂ produced a similarprofile of products from CO and H₂.

The inventors isolated microorganisms that could produce a highconcentration of ethanol from CO₂ and H₂ or from CO and H₂. For example,several isolates were shown to produce ethanol in media with greaterthan 10% ethanol by volume under conditions that favor ethanolproduction. These same isolates also produced ethanol when ethanol wasnot present in the fermentation medium. Surprisingly, when isolates thatproduce both acetic acid and ethanol were incubated with 6% or 10%ethanol, additional ethanol was produced at a faster rate and as agreater fraction of products (e.g. ethanol over VFA) than when the sameorganisms were incubated without ethanol at the start. It appears thathigh ethanol concentration actually inhibits production of acetic acidand other acids.

Some isolates convert a high percentage of synthesis gas to ethanolinstead of VFA, and under conditions described as an aspect of thisinvention can shift metabolism toward even greater ethanol production.Other isolates produce high concentrations of specific VFA. For example,one isolate produces 95% acetic acid (as a molar percentage of all VFA)from synthesis gases, while another isolate produced 48% butyrate, 6%propionate, and 6% isovaleric acid and less than 37% acetate even whenincubated under the exact same conditions as the isolate producing onlyacetic acid.

Most of these isolated organisms grow on glucose or other sugars as wellas on gases. Most isolates grew rapidly at pH 7 and continued to growwhen the pH was less than 5. Most isolated isolates could grow in mediawith VFA concentrations exceeding 3% by volume (e.g. 1% of each acetate,propionate and butyrate) at either pH (5 or 7). Some of the isolatescould tolerate or utilize O₂. The organisms generally grow rapidly andproduce the acids and alcohols under strictly anaerobic conditions. Theorganisms grew rapidly at 40° C., but also grew at lower temperaturesand higher temperatures such as 25° C. or 55° C.

Some isolated organisms could degrade biomass comprised of cellulose andhemicellulose to produce alcohols or acids and CO₂ and sometimes H₂.These same microorganisms took up CO₂ and H₂ released from the biomassdegradation to produce additional alcohol or acid. In some cases,microorganisms that produced mostly ethanol when digesting biomass wererecently discovered to also produce ethanol from CO₂ and H₂. Some ofthese microorganisms produced mostly ethanol, not other acids orproducts, and they continued to produce ethanol when the ethanolconcentration exceeded 7% of volume. Hydrogen gas may be added toincrease the use of CO₂ produced from biomass digestion that issubsequently assimilated into additional ethanol. For example, anorganism that produces ethanol from cellulosic biomass also producedCO₂. When H₂ was added to the fermentation, the organism assimilated theproduced CO₂ and the added H₂ into additional ethanol. Thus, more thantwo thirds of the carbon in the biomass was converted to ethanol.

In addition to using mono-cultures of microorganisms, mixed cultures canalso be used. In some cases, the isolates from the mixed culture mayhave included more than one isolate together (co-isolates), orseparately isolated organisms can be combined (co-cultures). Theproduction of certain products sometimes decreases when isolates arefurther purified by isolating colonies from the culture. It may be thatmore than one form share the needed activities. Nonetheless, themicrobial cultures can be transferred, maintained and grown for longperiods of time whether co-isolates, co-cultures or mixed cultures.

In addition to defined and isolated co-cultures, undefined mixedcultures may be used. The advantages of mixed cultures are that fewermicronutrients may be required because some isolates can transfernutrients (e.g. vitamins) to others, and different products may beproduced because a product may be exported from one isolate and taken upby another one. The advantage to undefined mixed cultures is thatsterile procedures would not be needed. Mixed cultures could come fromthe rumen of a cow, the hind but of an insect, compost or soils, amongmany other sources.

Sources of Microorganisms

The rumen microbial ecosystem, like many other microbial cultures,contains many microorganisms and pathways resulting in a broad array ofactivity. The end result of this activity normally is determined by thesecond law of thermodynamics, but the profile of products can be alteredby adding metabolites or inhibiting certain pathways. Further, the endproducts can also be manipulated by the complete removal of certainpathways by isolating specific organisms, using inhibitors withisolates, or genetically modifying organisms. The microorganisms can beundefined cultures or isolated cultures used individually or in mixedcultures to produce the desired activity for biofuel production. Forexample, ethanol can be produced by rumen microorganisms, but itnormally isn't because the organisms favor production of acids ratherthan ethanol under natural conditions. With different conditions,ethanol can be favored, and the organisms that produce it can beenriched and selected for.

Although any anaerobic fermentation may be an adequate source ofmicrobes, the rumen of a cow is especially advantageous. The rumen isconsistently warm favoring rapid metabolism and hosting 10¹⁰ organismsper ml. The high dilution rates wash out organisms that do not growquickly. These conditions are similar to anaerobic digesters to be usedfor the proposed process. The rumen gas phase is largely comprised ofcarbon dioxide and methane with enough hydrogen to make itthermodynamically feasible to produce carboxylic acids. Rumen microbesproduce several liters of H, per day and much more CO₂ and these gasesare transferred among microbial species and incorporated into methaneand carboxylic acids. The mixed culture rumen microorganisms are wellknown to produce acetic acid from CO₂ and H₂, and these microbes alsoconvert acetic acid to other volatile fatty acids by incorporating CO₂and H₂ to produce propionic acid, butyric acid, and longer chain acids.Rumen microbes are also well known for production of ethanol althoughethanol does not accumulate in the rumen because it is subsequentlyconverted to other products. Microbes may be removed from the rumen of acow by taking them directly from a fistula inserted into the cow's side.Additionally, microbes can be obtained using a stomach tube, or they maybe obtained from a slaughterhouse. Microbes may be taken from the fecesof a ruminant or from the hindgut as well.

There are many other sources of microorganisms that can be used in placeof microorganisms from the rumen of a cow. For example, microbes may betaken from the gut or feces of any other ruminant such as deer,antelope, bison, or camel. They may also be taken from elsewhere in adigestive tract of any type of animal including mammals or non-mammals.Even insects like carpenter ants or carpenter bees or termites hostlarge numbers of microbes that may be suitable. Microbes may be obtainedfrom soils, water bodies, compost, or manure digesters. Fermented foodsmay also host suitable organisms. For example, wine, cider and beer hostorganisms that are tolerant to acids and ethanol and that may use and/orproduce ethanol.

The following functions can be orchestrated by microbial culturesobtained from a mixed culture such as exist in the rumen of the cow andmany other natural and diverse ecosystems. These are functions that maynot be observed under natural conditions because of the need to limitsome activity that would naturally be present. For example, theproduction of biofuels or other desired products from synthesis gases,as opposed to production of acetic acid, is as much about limitingenzyme activity as it is about adding it. Isolation of microorganismscan often be used to limit the activity in the system because in naturethe metabolites are passed from one organism to the others, thus notproviding organisms to pick up the metabolite can enable a desiredmetabolite's accumulation. This application focuses on the way to effectthe following activities using microbial cultures, as well as ways todevelop the microbial cultures themselves:

-   -   a. Conversion of CO₂ and H₂ to acetic acid. The acetic acid can        be further converted to other VFA or alcohols with the same or        other cultures.    -   b. Conversion of CO₂ and H₂ to ethanol. The inventors discovered        and isolated organisms from the rumen of a cow that convert CO₂        and H₂ to ethanol.    -   c. The interconversion of acetic acid and ethanol. The direction        of interconversion would be controlled by thermodynamics.    -   d. Conversion of carbon monoxide (CO) and H₂ to acetic acid.        This process is a means to use gases produced from        high-temperature physical digestion. It is closely related and a        part of the pathway for the conversion of CO₂+H₂→acetic acid,        which is known to be predominant in the rumen. Wherein        CO+H₂O←→CO₂+H₂ is a rapid process at moderate temperature, the        system that assimilates CO₂ and H₂ into longer carbon chains        also assimilates CO and H₂. The inventors discovered that most        of the organisms that produce volatile fatty acids or lower        alkyl alcohols from CO₂ and H₂ produce a similar profile of end        products from CO and H₂.    -   e. Conversion of CO and H₂ to ethanol. Several organisms were        isolated from the rumen that could carry out this process.    -   f. The inventors also contemplate the direct conversion of CO₂,        CO₂ and H₂ to ethanol without producing acetic acid. Yeast are        known to make ethanol by two pathways that both pass through        acetic acid, and yet little acetate is released from the cell.        These pathways include: acetyl CoA to acetate to acetyl aldehyde        to ethanol and alternatively, pyruvate to acetate to acetyl CoA        to ethanol. The present application will show a set of        conditions that make it thermodynamically favorable to produce        ethanol from CO₂ and H₂ or CO and H₂ but not thermodynamically        favorable to produce acetate from the gases.    -   g. Conversion of organic acid to alkyl alcohol. For example,        lactic acid conversion to ethanol, or butyric acid conversion to        butanol.    -   h. Conversion of one organic acid to another. For example,        acetic acid converted to propionic acid, or acetic acids        converted to butyric acids, or further elongation of carboxylic        acids, or shortening of carboxylic acids.    -   i. Conversion of acetic acid to H₂ and CO₂, which could also        include acetic acid degrading organisms. These organisms would        be used in combination with a way to make acetic acid        degradation thermodynamically favorable such as by purging of        gases.

Often more than one function above would be combined. For example, onemicroorganism may use CO₂ and H₂ to produce acetic acid (function a),while another one may convert the acetic acid to ethanol or butanol(function c). This combination could result in production oflonger-chain alcohols from CO₂, CO and H₂.

A natural anaerobic or aerobic ecosystem has the enzymes to produce manydifferent products or intermediates, but typically these systems arestable and produce the same profile of products. By selecting forcertain enzyme activity and removing others, the desired products can beproduced and concentrated.

An efficient way to make acetic acid from waste gases is to maintain apopulation of reductive acetogens (function a) with high enoughpressures of CO₂ and H₂. Others have proposed adding reductive acetogensto increase acetic acid production in the rumen and decrease methanelosses. However, this method does not work in the cow's rumen becausethe acetogens are already there, and rumen acetate is near equilibriumwith CO₂ and H₂. On the other hand, a digester can increase its totalpressure, or the partial pressure of either or both gases to promote thereaction. A culture that does not contain methanogens would naturallymake it possible to maintain higher H₂ and CO₂ pressure than one thatcontains methanogens, and therefore removing or inhibiting methanogenspromotes greater synthesis of carboxylic acids or alcohols from CO₂ andH₂.

Because the rumen has the microorganisms to assimilate CO₂ and H₂ intoacetic acid, and acetic acid is in equilibrium with ethanol, thepathways are present to convert CO₂ and H₂ to ethanol.

Desired Characteristics of Microorganisms to Assimilate Synthesis Gases

An aspect of the present invention is the means to obtain pure culturesof microorganisms that can tolerate high concentrations of alkylalcohols and acids while they produce additional alcohols or acids.

The present application describes the means to enrich for or selectmicroorganisms that can be used to produce products synthesized fromgases. The first step to synthesize or enrich for these organisms is toidentify the desired physiological traits for the organisms.Understanding these desired traits enables the establishment ofconditions where the organisms with those traits thrive

The ideal characteristics of desired organisms are as follows:

-   -   Produce desired product or products from gases (CO, CO₂, H₂)    -   Produce desired product or products nearly exclusively    -   Produce desired product or products to a high concentration    -   Produce desired product or products at a high rate

In this case, the desired product may be acetic acid or ethanol, or amicrobial culture may be developed to produce a longer chain alcohol oracid from gases, such as propionate or butyrate, or 1-propanol or1-butanol. For example, microorganisms in the rumen have been shown tointerconvert acids like acetic acid, propionic acid, and butyric acid,and to interconvert acids with alcohols. The extent to which theseinterconversions occur depends on the thermodynamics and theconcentrations of various metabolites for the desired pathways as wellas competing pathways. Thus, the enzyme activity exists in rumen fluidto convert gases to acetic acid, ethanol, propionic acid, propanol,butyric acid, butanol as well as many other acids and alcohols. The factthat a product occurs in rumen fluid, even at low concentration, impliesthat at least one isolate of microorganism produces the product. Thepresent patent application describes the means to isolate the desiredactivity so as to produce the product desired instead of mostlyproducing other products.

Whatever the product or products desired, the desired cultures ofmicroorganisms should be limited in their ability to produce non-desiredproducts. For example, when using rumen fluid as the microbial culture,under the correct conditions any of several desired products can beproduced from CO₂, CO and H₂, but several different products will resultwhich together may be difficult to separate or may not be desired at agiven time. The gases may produce acetic acid and ethanol but these maybe interconverted with propionate and butyrate and propanol and butanol.A mixed culture can be controlled to some extent by controlling thefermentation gas mixture and other aspects of concentrations that affectthermodynamics, but several products are likely to be feasible and withtoo many reaction pathways available it becomes difficult to controlwhich products are produced. In contrast, an isolated microorganism thatonly produces acetic acid and ethanol can be controlled to some extentby thermodynamics (e.g. higher H₂ partial pressure and lower pH increasealcohol over the acid).

If a microorganism is selected that only produces a certain desiredproduct under a specific set of conditions, it will only be able tosurvive when the thermodynamic conditions are such that production ofthe desired product will enable it to obtain energy. When theconcentration of product is increased, the thermodynamic equilibrium canshift against greater production of the product. The organism maysurvive by 1) producing a different product, or 2) catabolism(breakdown) of the desired product. It may appear that the microorganismis intolerant to the desired product. However, increasing theconcentrations of substrates, decreasing the concentration of otherproducts, or adjusting the ratios of products or substrates can shiftthe metabolism toward greater product production at even greaterconcentration of the product. Often, the appearance of intolerance to aproduct can be overcome this way. One aspect of the present invention isto overcome these apparent intolerances by readjusting products orreactants, especially gas pressures.

At some concentration of product, such as ethanol or acid, themicroorganism that produces the product is in deed intolerant to thefurther concentration of the product even after adjusting for the otherproduct and reactant concentrations. For example, enzymes or cellmembranes may be inhibited by the product. Another aspect of the presentinvention includes isolation and identification of microorganisms thatare tolerant to high concentrations of the desired products: ethanol,propanol, butanol, acetic acid, propionic acid, or butyric acid, otheralcohol or acid, or a combination thereof. The process to isolate themicroorganisms themselves with high tolerance to their products, and themethods to isolate them are both aspects of the invention.

The organisms that comprise an aspect of the present invention are alsoable to produce the desired products at high rates. The organisms areisolated and screened for such high rates as an aspect of the presentinvention.

Other characteristics of the microorganisms that may be desired but notrequired include characteristics that make them easier to handle or moreversatile including:

-   -   Ability to utilize or tolerate oxygen    -   Ability to digest biomass including cellulosic biomass

Some microorganisms isolated can digest cellulosic biomass as well assynthesize ethanol from CO₂ and H₂. Methods disclosed by the inventor inU.S. Ser. No. 61/113,337, which is incorporated by reference were usedto isolate organisms that produced ethanol from cellulosic biomass. Insome cases, these microorganisms appeared to convert more than 67% ofthe cellobiose to ethanol, sometimes appearing to convert nearly all ofthe cellobiose carbon to ethanol when incubated with 2 atm H₂. Theinventors expected the maximal conversion of carbon to ethanol to be 67%with known pathways producing 2 ethanol per glucose equivalent, and 2CO₂. At the same time, the total gas pressure was found to decreaseduring the incubation, and the acetic acid concentration sometimesdecreased. In one case, the ethanol concentration increased above 7.5%by volume. Thus, conditions for isolation and incubation ofmicroorganisms that were intended to obtain fiber-digesting microbesthat could produce ethanol, in some cases, resulted in isolation ofmicroorganisms that could also produce ethanol from CO₂ (produced in thefermentation) and added H₂. The results were not expected, and in factthey were dismissed until they were found repeatedly for certainmicroorganisms.

The inventors isolated microorganisms that can assimilate CO₂, CO and H₂into ethanol while the concentration of ethanol exceeds 10% by volume.The thermodynamic analysis also suggests that it is feasible to producehigh concentrations of ethanol from high CO₂, CO and H₂ concentrations.At low pressures of these gases, or if the ratio is skewed against thecarbon or hydrogen side, it is thermodynamically more favorable todegrade ethanol than to produce it.

The inventors also contemplate microorganisms, and the isolation ofmicroorganisms that can produce propanol or butanol from CO₂, CO and H₂.The fact that ruminal microorganisms can assimilate acetic acid from CO₂and H₂ has been previously reported. The acetic acid is in equilibriumwith other acids and alcohols in mixed culture fermentations, includingthe fermentation in the rumen. Thus, the enzymes and pathways exist forthe ultimate production of any of these products from CO₂, CO and H₂.Microorganisms were isolated from rumen fluid that were tolerant to asmuch as 6% butanol and which produced butanol from cellulosic biomass.Therefore, pathways are present in rumen microorganisms to producebutanol from biomass, and since these pathways are linked, the entirepathway from gases to butanol is present. The butanol-producingorganisms were tolerant to as much as 6% butanol, so producing butanolfrom CO₂, CO and H₂ at a similar concentration was contemplated. Theability to also produce propanol at high concentration from CO₂, CO andH₂ was also logically contemplated similarly.

The inventors discovered that when incubating mixed cultures of rumenbacteria with as much as 6% 1-propanol, 6%1-butanol, or 10% ethanolunder either aerobic or N₂ gas phase, the fermentation activelyproceeded producing gas from the alcohols. The same enzymes tocatabolize alkyl alcohols would be used to synthesize them under CO₂, COand H₂ pressures, and these enzymes are present in rumen fluid inorganisms tolerant to the alcohols. Organisms that survived these highconcentrations of alcohols were found to also produce the alcohols athigh concentrations under conditions favoring synthesis of thesecompounds.

Isolation of Microorganisms

Another aspect of the invention is the discovery that many mixed culturefermentation systems, including the rumen of cattle, have all of thedesired activity to produce the desired product. In order to convert ahigh percentage of the gas to one or more desired products (e.g. ethanolor butanol), the undesired pathways (e.g. acetic acid) need to beprevented. Various inhibitors can be used to slow or shut down undesiredpathways. In addition, concentrations of products and reactants can beadjusted to make the desired pathways more favored over the undesiredones. However, when there are many different pathways in a singleculture, it can be difficult to end up with only one or two products.Shutting down one pathway often opens another one. A way to limit thenumber of pathways in the fermentation is to isolate microorganisms withfew pathways present from the mixed culture. The various methods used toinhibit undesired pathways (inhibitors or thermodynamics) can be used inthe isolation process.

One aspect of the process is to use conditions to control thefermentation to isolate microorganisms that comprise an aspect of theinvention. For example, adjustments in gas pressures can be used toenrich and select for microorganisms that are able to produce greaterconcentrations of desired products (e.g. a specific alcohol or acid) andconvert more of the gases to that desired product. Using inhibitors toselect against acetic acid production, for example, can also be used toselect against acetic acid producers. In this way, organisms can beisolated that produce a high percentage of the desired product, such asethanol or 1-propanol, because they do not have pathways to produceother products.

The microorganisms that can be isolated to assimilate alcohols and VFAfrom synthesis gases (CO₂, CO, H₂) and that would be tolerant to theproducts they produce, are disclosed as an aspect of this application.These microorganisms continue producing the products even when theconcentration is high. The way to manipulate other metabolites to makethe high concentrations thermodynamically favorable, and undesiredproducts unfeasible has been disclosed as an aspect of this invention.Using those same conditions to enrich, select and isolate microorganismsthat are tolerant to the products is another aspect of the invention.For example, microorganisms can be isolated in the presence of highconcentrations of 1-butanol or 1-propanol, and with thermodynamicconditions to synthesize these alcohols from gases (i.e. high partialpressures of the gases at a ratio of 3 to 1 of H₂ to CO₂). It isthermodynamically feasible to make more of the alcohols even in thoseconcentrations so organisms that carry out the reaction are favored.Alternatively, for a step in the process, microorganisms that utilizethe 1-propanol or 1-butanol might be selected by incubating in highconcentrations of the respective alcohol and gases that favordegradation of the alcohol (i.e. low CO₂ and/or H₂). In a subsequentstep, these microorganisms can be selected under high CO₂ and H₂ andoptimal ratios so that the organisms that can obtain energy from theopposite pathway are selected.

In general, the unique aspects of the microbial isolation methods formicroorganisms that are especially suited for synthesis of alcohols oracids from synthesis gases that are an aspect of the present inventionare as follows:

-   -   Isolation with high concentrations of the product    -   Isolation under conditions that thermodynamically favor the        product formation    -   Isolation under conditions that disfavor competing pathways    -   Thermodynamically disfavor undesired pathways    -   Inhibitors disfavor undesired pathways    -   Isolation that favors microbes that grow quickly or produce        products at high rates

For example, ethanol-tolerant microorganisms that produce ethanol fromsynthesis gases were selected by growing a mixed culture for manygenerations in the presence of the gas concentrations that favor ethanolproducers more than acetic acid producers. Acetic acid was furtherinhibited in some cases by growing the cultures at pH less than 5. Afterenrichment, the culture was diluted to isolate individual colonies thatgrow from gases as the only energy source (in a ratio favoring ethanol,3:1 of H₂ to CO₂), and which may include ethanol in the media. With adifferent gas mixture, ethanol would be degraded but calculations showedthat it would be thermodynamically infeasible to degrade it, even athigh concentrations, at high partial pressures and the ratio of 3:1 ofH₂:CO₂. The same process can be applied to carboxylic acids and otherproducts.

Example Process to Isolate Microorganisms that Synthesize Alcohols fromGases

Several different sets of conditions were used to select formicroorganisms that could produce alcohols from CO₂, CO and H₂. In oneapproach, conditions that result in degradation of the desired alcoholsselect for organisms that can also synthesize the desired alcohols oracids. Thus, in some cases conditions were established to enrich orisolate the degraders of products rather than the producers of theproducts directly. This enabled more specific selection of organismsthat were tolerant to a certain product and which had the pathway tomake the certain product. For example, microorganisms that degraded1-butanol to CO₂ and H₂ also had the enzymes to make 1-butanol from CO₂and H₂. On the other hand, an organism that uses CO₂ and H₂ could makemany different products including other alcohols and acids and may notbe ethanol tolerant.

Some enrichment and isolation (roll tube) conditions used to enrich andisolate organisms to produce ethanol, propanol or butanol from CO₂, COand H₂ are shown in Table 2. For isolations numbered 1 to 6, theenrichment selected for microorganisms that degrade the respectivealcohol to gas. Using N₂ in the gas phase and high concentrations of thealcohols thermodynamically favored the degradation of the alcohols.Other gases were used in place of N₂, including air, CO₂ or H₂. In thecase of air, aerobic or aerotolerant microorganisms were selected. Thesewere easier to handle in some applications. In the case of CO₂ and H₂,degradation of the alcohols was still favored thermodynamically becauseof the high or low ratio of H₂:CO₂ or H₂:CO. Then, isolates wereselected that could grow on the gases which were provided inconcentrations to thermodynamically favor alcohol production.

Alternatively, in isolations numbered 7 to 8, organisms that could growon CO₂, CO and H₂ were enriched, selected and isolated. These organismsproduced a number of different products, which were determined throughanalysis on a gas chromatograph or other procedure. Using a ratio ofH₂:CO₂ of 3:1, pressure of 2 to 4 atm and potentially moderately low pH(e.g. 4-5) favor alcohols over acids. This combination of conditions canbe used to shift fermentation toward alcohols. Previous investigatorsdid not isolate organisms under conditions that made the desired alcoholor carboxylic acid thermodynamically feasible, and especially notthermodynamically favorable compared to other potential products.

In addition to the conditions shown in Table 2, other conditions alsowork well. For example, the ratio of H₂ to CO₂ could be higher, such as10 to 1 or 100 to 1 while it is still feasible to produce some ethanolrather than degrade it. This higher ratio shifts fermentation further infavor of alcohols or longer-chain carboxylic acids over acetic acid, andconditions can be established in which it is only feasible to makeethanol and not make acids. Many of these enrichments were undertakenwith 2 to 4 atm total pressure to further shift the fermentation towardalcohols and longer-chain acids. It is also advantageous to includealcohols in the enrichment or in the isolation medium and when the ratioof H₂ to CO₂ or H₂ to CO is near the optimal level, and especially whenunder greater than 2 atm and preferably greater than 4 atm totalpressure. It is still thermodynamically feasible to produce more of theethanol or desired acid. This inclusion increases selection pressure toobtain more alcohol-tolerant organisms. It is also advantageous toinclude acetic acid or other VFA in the media during enrichment orisolation as the VFA inclusion shifts equilibrium against further VFAproduction and also selects for organisms that are tolerant to higherconcentrations of VFA. Individual VFA or a mixture of several were used.Some organisms may be intolerant to the VFA and would be selectedagainst, while others can grow in the presence of VFA. These isolationswere conducted at pH 7 or pH 5 with or without 3% mixed VFA. Isolateswere also obtained at pH 4, and higher or lower pH could also be used.Some organisms could be isolated at higher VFA mixtures, such as 6%total VFA or 10% VFA. The lower pH and the aerobic conditions selectedfor organisms that produced greater molar ratios of ethanol to aceticacid even when incubated at pH 7 under similar conditions following theisolation.

TABLE 2 Exemplary Enrichment and Isolation Conditions to ObtainMicroorganisms that Synthesize Alcohols to High Concentrations.Enrichment Isolation No. Alcohol Gas Mix Alcohol Gas Mix 1  6% EthanolN₂, air, H₂, or CO₂ 0 3:1 H₂:CO₂ or 2:1 H₂:CO 2 10% Ethanol N₂, air, H₂,or CO₂ 0 3:1 H₂:CO₂ or 2:1 H₂:CO 3  4% 1-Butanol N₂, air, H₂, or CO₂ 03:1 H₂:CO₂ or 2:1 H₂:CO 4  6% 1-Butanol N₂, air, H₂, or CO₂ 0 3:1 H₂:CO₂or 2:1 H₂:CO 5  3% 1-Propanol N₂, air, H₂, or CO₂ 0 3:1 H₂:CO₂ or 2:1H₂:CO 6  6% 1-Propanol N₂, air, H₂, or CO₂ 0 3:1 H₂:CO₂ or 2:1 H₂:CO 7 03:1 H₂:CO₂ 0 3:1 H₂:CO₂ 8 0 2:1 H₂:CO 0 2:1 H₂:CO

Medium for enrichment, isolation, screening and initial experiments wasas described for in vitro digestion according to the manual by H. K.Goering and P. J. Van Soest, 1970. (Agricultural Handbook No. 379entitled Forage Fiber Analyses (Apparatus, Reagents, Procedures, andSome Applications, Agricultural Research Service of the United StatesDepartment of Agriculture), which is incorporated by reference, exceptcarbonate and bicarbonate salts (e.g. NaHCO₃, NH₄HCO₃) were replacedwith equi-molar phosphate buffer salts (e.g. NaH₂PO₄, NH₄H₂PO₄) adjustedto pH 7 or pH 5 unless otherwise indicated. Macro minerals (e.g.calcium, magnesium), microminerals (e.g. iron), tryptic digest ofcasein, ammonia, sulfide and cysteine reducing agents, and resazurinwere used as described by Goering and Van Soest (as cited). Media wereboiled under 1 atm N₂ gas. The same media formulae were used for initialenrichment, agar roll tubes, broths, slant tubes, and agar plates.However, roll tubes, slant tubes and agar plates also contained 2% agar(BactoAgar). Media used for pure cultures (everything but the initialenrichment) were combined with 20% autoclaved rumen fluid to providepotential unknown growth factors. For some enrichments and isolations,some nutrients were omitted to select for organisms that do not requirethese nutrients or can synthesize their own. For example, ammonia may beomitted and amino acids used instead, or the other way around to selectfor organisms that synthesize protein from ammonia or degrade proteinand use amino acids.

Enrichment.

Media usually minimized extraneous sources of energy (e.g. glucose), toselect for organisms that could grow from the energy they captured fromthe gases or from the alcohol added. Medium (45 ml) was transferred toeach flask. Rumen fluid was collected from the cow's rumen through afistula and was initially prepared by blending for 1 minute andstraining though cheese cloth followed by glass wool. Carbon dioxide,run through a copper column to remove O₂, was perfused over rumencontents and into containers to maintain anaerobic conditions. Rumeninocula (5 ml) was added to each 250-ml flask. Large flasks relative tothe volume of liquid were used so the headspace composition of gases didnot readily change. Flasks were sealed with butyl rubber stoppers, andif needed vacuum was applied to remove initial head space gas beforeperfusion with corresponding treatment gases using a needle and syringe.Cultures were incubated in a shaking water bath for 3 days, and newgases were perfused at least daily. New flasks were prepared with 45 mlof media, and 5 ml of subculture was added from the previousfermentation. These flasks were again incubated for 3 days, and againsub-sampled. Each enrichment process included several cycles ofsub-culturing and growth. Many variations on enrichment cultureconditions are acceptable or advantageous, and can be applied.

For each enrichment and roll tube, the temperature of incubation was 39°C. unless indicated otherwise. Organisms were also selected at 55° C.and other temperatures could have been used. Using differenttemperatures selects for organisms that thrive at differenttemperatures. Initially, various concentrations of alcohols were used.These conditions selected for organisms that could utilize the alcohols,and which therefore also had the enzymes to make the alcohols. The samecan be done for specific carboxylic acids. Each enrichment and isolationwas attempted with media buffered to pH 7, pH 5, or pH 4. The lower pHfavored alcohol producers and the isolates that resulted produced agreater concentration of ethanol and other alcohols relative to aceticacids and other volatile fatty acid production.

Isolation.

Roll tubes and agar plates used the same medium as for enrichment butalso contained 20% strained and autoclaved rumen fluid formicronutrients. The prepared medium (9 ml) was transferred to steriletest tubes while still hot from boiling Tubes were cooled to 55° C., andinoculated. Subcultures from the last enrichment were diluted seriallyin media to obtain cultures from 1 to 10′ viable cells per 0.5-mlinocula. Roll tubes were prepared for each level of dilution, butconcentrating on the 10⁻¹⁰ to 10⁻¹⁴ dilutions. Once inoculated, tubeswere perfused with gas, stoppered, and rolled on ice to make the gelharden. Tubes were incubated for 24 to 48 h at 39° C. and independentcolonies selected from these. Only organisms that could grow using thegases as an energy source were selected in the roll tubes, in thepresent case, and pH indicators in the roll tube agar were used toidentify acid-producing and non-acid-producing isolates. In laterisolations, agar plates were incubated in 32-liter pressure cookersunder the desired gas pressures. These anaerobic chambers were easier tomanipulate en masse than individual tubes. Some plates were buffered topH 4, 5 or 7 and sometimes VFA were included.

Maintenance.

Colonies were selected from among the colonies in roll tubes and agarplates, and were transferred to broth for short-term maintenance. Thebroth was of the same composition as media for other purposes, but didnot contain agar. All cultures were maintained in large tubes leaving ahigh proportion of headspace, the ratio of CO₂ to H₂ or CO to H₂ thatfavors synthesis of the alcohols, and gas pressures of 2 atm to 4 atm.Broth cultures were maintained at 39° C. until they become cloudy, andnew gas mixes were perfused if necessary. Broth cultures (5% finalconcentration, vol/vol) were transferred to new media one to two timesper week. After two to three cultures, 0.1 ml culture was transferred toa slant culture in a 25-ml tube with 10 ml media on a slant to increasesurface area). The inocula were added on top of the agar, which wasmaintained under CO₂ and H₂ or CO and H₂ in the thermodynamicallyfavored ratio. These were initially incubated at 39° C. until coloniesformed (16 h), and then stored at 25° C. or 4° C. for up to a month.Cloudy broth cultures were also stored by adding 15% glycerol (finalvolume) and freezing in liquid nitrogen; once frozen, these colonieswere stored at −80° C.

Screening.

Screening of microorganisms addresses whether they can synthesize acertain alcohol or acid from CO₂, CO₂ and H₂, and the extent to whichthey are tolerant to the product. The isolates derived as described werescreened by transferring 0.5 ml broth to 9.5 ml media (as described, noagar). Tubes were perfused with mixtures of CO₂, CO and H₂ to favorsynthesis (e.g. 3:1 ratio of H₂:CO₂) of the desired product preferablyunder at least 2 atm total gas pressure. Preferably 4 atm total gaspressures are used. However, successful isolations were also performedwith only 1 atm total gas pressure with the ideal ratio of gases. The pHof the media was adjusted to 7, 5, or 4 and 3% or 6% mixed VFA added fordifferent runs. Samples were also incubated with and without initiallyincluding 6% or other concentration of an alkyl alcohol in the media.The cell growth was determined by turbidity, and alcohols and acids weremeasured by gas chromatograph at time=0, and other time points (e.g. 3 dand 5 d). All colonies were typically screened after first isolatingthem by adding the colony directly to a test tube with media, incubatingwith H₂ and CO₂ and determining which isolates produce the highestconcentrations of desired products or show other desired traits. Theisolates that appeared to be most ideal were sub-cultured and incubatedagain in fresh media in replicate to verify results and test for effectof different conditions (e.g. pH, gas pressure) of the fermentation.

Improving Organisms that Synthesize Alcohols or Acids

These same conditions to enrich or isolate microorganisms that producehigh concentrations of alkyl alcohols or acids from CO₂, CO and H₂ wereused to improve the isolated microorganisms. Some improvements occur inthe enrichment or isolation process although the improvements may not beobserved. Pure cultures of microorganisms can be incubated with theratio and pressure of gases that thermodynamically favors synthesis ofthe desired alcohol or acid, under pressures (e.g. 2 to 4 atm orhigher), and in the presence of the products to which tolerance isdesired. Under these conditions, organisms that produce the most of thedesired product thrive, while those that produce more of the undesiredproduct waste energy and become diluted. Over many generations, whichcan occur in a matter of days, organisms evolve under these conditionsthat can synthesize greater quantities of the desired product relativeto other products, at faster rates, and that are more tolerant of theproduct and potential co-products. Mutation rate can be increased bybrief exposure to UV light or other mutagen combined with thermodynamiccontrols. Using these conditions and several subcultures for enrichmentof pure cultures, organisms can be developed that make only the desiredproducts, at high rates, with high tolerance to those products. Thisprocess of directing evolution (or non-specific mutagenesis) selects fororganisms on the basis of the products they make, and selects fororganisms that can tolerate very high concentrations of products.

Previous attempts at non-specific mutagenesis did not select fororganisms that produced specific products, so one could not increase theamount of those products produced as a portion of total products. As aresult, many different products were made. In addition, previous methodsat adaptation to higher levels of products (e.g. alcohols) by growingthe isolates with the products selected against further production ofthose products (because they became thermodynamically limited). Bymaintaining highly thermodynamic ally favorable conditions for theproduction of desired products (e.g. alcohols) organisms are adaptedthat produce the desired products even at high concentrations. Usingthis process, the inventors contemplate isolating organisms that canproduce a desired alcohol or desired carboxylic acid nearly exclusively,and at very high concentration. For example, some natural organismsalready isolated produced virtually nothing but ethanol at greater than10% ethanol concentration by volume, but with use of the methods ofmutagenesis (or directed evolution) described in this application, theethanol concentration could approach 20% or more. Organisms could beadapted to produce virtually nothing but the desired alcohol at anyconcentration between 0 to 20% lower alkyl alcohol by volume.

SPECIFIC EXAMPLES Example Enrichment and Isolation of Microbes thatProduce Carboxylic Acids and Alcohols

In one example experiment, organisms were isolated by the proceduresdescribed in this application. The pH for enrichment and isolation was7. The incubation temperature was 39° C. Twelve enrichment treatmentswere used in a 6×2 factorial arrangement, with inclusion of one of 6alcohols with N₂ or air. Media for the enrichment contained one of thefollowing alcohols: 6 or 10% ethanol, 4 or 6% 1-propanol, or 4 or 6%1-butanol. The gas phase during enrichment was N₂ or air at 1atmosphere. After enrichment, agar for isolation did not containalcohols (removed substrate) but headspace gas was 3:1H₂ to CO₂ at 1 atmin roll tubes.

Microorganisms grew in all enrichment treatments, and isolates wereobtained from all treatments except for 6% 1-butanol. A total of 127organisms were isolated based on their ability to grow on agar with H₂and CO₂, and each isolate was screened by transferring the colonies tobroth (as described) and incubating for 5 days under 1 atm gas pressureof 3:1H₂ to CO₂ without shaking (although higher pressure and shakingmay be preferred). Each isolate was incubated in a sealed tube with 5 mlof media and 10 ml of headspace gas.

Most of the isolates did not grow on synthesis gases or did not producesignificant amounts of alcohol or VFA in broth after isolating thecolonies. Using only 1 atm pressure may not have strongly favoredmicrobes that grow on synthesis gas over those using residual energy inthe media. However, several isolates did grow as indicated by increasedturbidity, production of VFA or ethanol, and a decrease in headspace gaspressure. The 16 colonies that produced the most interesting profile ofproducts (as measured by gas chromatography) were sub-sampled and againincubated in duplicate under similar conditions.

The concentrations of VFA and alcohols were measured after 5 days and 10days. However, after 5 days about half of the headspace gas wasincorporated into VFA or alcohols and growth did not continue. Inaddition, duplicate samples of each isolate were incubated with N₂ gasin the headspace instead of H₂ and CO₂ and under otherwise identicalconditions, but cells did not grow or did not grow as much in thosecases and did not produce as much VFA or ethanol. This negative controlconfirms that the organisms were growing from the H₂ and CO₂ gas and theenergy from making VFA and alcohols. Example VFA profiles for organismsthat produced VFA from synthesis gases are shown in Table 3. The resultsare for the mean of the duplicate samples from separate fermentations ofthe same isolate, which produced similar amounts of each product.

TABLE 3 Product profile of selected isolated isolates of microortanisms.Ace- Propi- Buty- Iso- Valer- Iso- Etha- tate onate rate Butyrate ateValerate nol Isolate Molar % of Total VFA S3 56 10 34 0 1 0 3 S13 36 648 2 2 6 1 S90 95 2 0 0 2 1 3 S99 46 4 2 18 4 27 7 S120 42 2 1 20 1. 3420 S121 45 5 4 18 1 27 15Mean standard error=0.07%, n=30. Molar percentage means the molar ratioas a percentage of total VFA times 100. Molar ratio means theconcentration of the individual VFA or ethanol produced divided by theconcentration of total VFA measured.

Isolates S3 and S 13 were isolated after enrichment in 6% ethanol.Isolates S90 and S99 were isolated after enrichment in 4% propanol, andisolates S120 and S121 were isolated after enrichment in 4% butanol.

The results indicate a tremendous variation in which products theorganisms could produce when incubated under similar conditions. IsolateS90 produced mostly acetate and small amounts of ethanol. Isolates S3and S13 produced a high percentage of butyrate, and strains S99, S 120,and S121 produced large amounts of iso-butyrate and iso-valerate. Oneisolate produced about 12% molar percentage as valerate but theduplicate did not grow so results are not shown.

Although some isolates only produced a small amount of alcohols,isolates S120 and S121 produced important concentrations of ethanol whenincubated under the same conditions as other products. These isolatesalso produced 1-propanol, iso-propanol and 1-butanol. Profiles ofalcohols were similar for both isolates, which produced 11.4 or 11.0molar % as much 1-propanol as total VFA, and 3.3 or 2.8 molar % as muchiso-propanol, and 2.9 or 2.8 molar % as much 1-butanol as total VFA forstains S120 or S121 respectively.

Wherein others have isolated organisms that could synthesize acetate ortrace quantities of ethanol, the present results show that using theisolation techniques described in this application, organisms could beisolated that preferentially produced alcohols, including 1-propanol,and VFA that are seldom observed from synthesis gases, such aspropionate, butyrate, iso-valerate, and iso-butyrate.

Animal nutritionists believed previously that branched chain VFA(iso-butyrate and iso-valerate) in the rumen were derived exclusivelyfrom amino acids in the feed, and adding them to the rations ofruminants increases animal growth and milk production, particularly forlow-protein diets. The quantity of these VFA produced from synthesis gasby some of these isolates were orders of magnitude higher than thequantity of branched-chain amino acids in the media, and they were notproduced in corresponding tubes with N₂ in the headspace instead of H₂and CO₂. Thus, these VFA were produced by a previously-unknown process.These particular VFA are valuable as feeds for animals, includingruminants. The organisms themselves may be used to enhance digestion andmetabolism in the gut.

Example Effect of Enrichment pH

In one set of isolations, the effect of pH and inclusion of VFA in themedia was tested for the enrichment phase otherwise as describedpreviously. Enrichment treatments were compared in a factorial designwith 2 pH levels (5 or 7), inclusion of VFA (1% of each: acetate,propionate, and butyrate) or not, for media with one of the alcohols (6%ethanol, 4% 1-butanol, or 4% 1-propanol), using N₂ or open air as thegas phase. Rumen fluid was used as the inocula which was incubated at39° C. After enrichment and sub-culturing 3 times as described, cultureswere isolated as described by incubating various dilutions on agarplates incubated in an anaerobic chamber with 2 atmospheres of gas with3:1 ratio of H₂ to CO₂. Cultures grew under each enrichment condition,and isolates were obtained that grew on the synthesis gases for eachenrichment treatment except no isolates were obtained from theenrichment in butanol under aerobic conditions (isolates were obtainedfrom enrichment in 1-butanol under N₂).

Isolates were screened by incubating in broth at pH 7 under 2 atmpressure of 3:1 of H₂ to CO₂. Most produced low quantities of ethanol oralcohols, but several produced more than 20% as much ethanol as totalVFA. Most of the isolates from this run grew on synthesis gases, but themain difference for the pH 7 treatment without added VFA compared to thelast enrichment was the use of 2 atm pressure for both enrichment andscreening. The higher pressures would greatly favor synthesis gas usersduring isolation and enable more of them to obtain energy from synthesisgas during screening. The pressure would also favor alcohols andlonger-chain VFA.

The product profiles from some isolates are shown in Table 4. A total of25 organisms (of 300 isolated in this run) produced a molar ratio ofethanol to acetate greater than 0.4. This means the concentration ofproduced ethanol in moles per liter divided by the concentration ofproduced acetate in moles per liter was greater than 0.4. One isolate(S241) produced a molar ratio of ethanol to acetate of 0.89, and one(S202) produced a ratio of 0.70. The molar ratio of ethanol to all VFAwas 0.43 and 0.37 (mol/mol) respectively. These two isolates alsoproduced other alcohols with the predominant alcohol after ethanol being1-butanol. The isolates S241 and S202 also produced butyrate andpropionate.

The low-pH enrichment in the present experiment provided a higherpercentage of organisms producing alcohols and higher ratios of alcoholsto other products. At low pH, the thermodynamics disfavors acidproduction and it also selects for alcohol producers. In addition,organisms that were enriched in air produced more alcohol when screenedthan organisms enriched under N₂.

TABLE 4 Product Profiles from Example Isolated Isolates. AcetatePropionate Butyrate Ethanol Isolate Enrichment molar percentage S241Media with VFA 48.0 8.9 39.1 43.0 and 6% ethanol, pH 5 in N₂ S202 Mediawith 6% 53.4 10.5 31.5 37.3 ethanol, pH 5, in N₂ S392 Media with 48.240.1 8.0 7.3 VFA and ethanol, pH 7 in air S247 Media with VFA 34.3 14.049.4 9.7 and 6% ethanol, pH 7 in N₂Molar percentage as a fraction of all measured VFA (acetate, propionate,butyrate, iso-butyrate, valerate, iso-valerate). Molar percentage=molarratio times 100.

Microbial Characterization and Identification

All 32 isolates from the above isolation process were comparedmorphologically for differences. All isolates formed visible colonies onagar from synthesis gas within 5 days, and on glucose agar within 1 day.Colonies on agar were round, and yellow or white in color. All but oneof the 32 isolates grew on glucose media in open air. Most of theisolates tested positive for gram stain, and were spherical cocciwithout spores present. Some isolates presented mostly as individuals,some were found in doublets, and other isolates were found in chains.Most of these isolates were negative to catalase test or slightlypositive. One isolate was strongly catalase positive. The one organismthat did not grow in open air, did grow on glucose media under strictanaerobic conditions. It was a spiral-shaped, gram-negative organism.Based on these characteristics, organisms appeared to be from severaldifferent isolates, especially from the genus Enterococcus and/orStreptococcus among others.

One of the best producers of ethanol and butyrate (S241) was furtheridentified. Gene sequencing of 500 base pairs (bp) in a region of 16SrRNA identified this organism as having 99.89% homology (1 bp differed)with Enterococcus avium. Greater than 97% homology was also observedwith E. gilvus (99.84), E. malodoratus (98.63), E. pseudoavium (98.21),and E. Raffinosus (97.89). Although the organism may not be of the samespecies as any of these, less than 97% homology is generally used todenote a different species. In other words, based on the geneticsequence alone, one cannot rule out that the organism is a member of oneof the species listed. However, the ability to produce highconcentrations of ethanol or butyrate from synthesis gases was not knownfor any member of the genus Enterococcus, or for any other facultativeanaerobe.

The diversity of the organisms that were isolated shows that it ispossible to isolate many different species of organism using thetechniques described, and many different species harbor the traitsmaking it possible for them to produce alcohols or carboxylic acids fromsynthesis gases. Many of the isolates would be robust and easilymaintained and used in a scaled-up procedure. They grew quickly andcould be maintained or grown on glucose or synthesis gases, and wereaerotolerant.

Example Comparison of CO and CO₂

All 32 stains from the preceding experiment were also incubated with a2:1 ratio of H₂ to CO compared to a 3:1 ratio of H₂ to CO₂. Otherconditions were similar to previous incubations including 2 atm totalpressure, initial pH of 7, temperature of 39° C. Each isolate wasincubated in duplicate fermentation tubes for each set of gas.Concentrations and profiles of products were similar across mostisolates. One isolate produced more acetate and less propionate andbutyrate with CO compared to CO₂. Two isolates did the opposite andproduced more acetate and butyrate but less propionate with CO comparedwith CO₂. The results indicate the approach can be used for both CO andCO₂ with equal effect for most isolates.

Example of Controlling Production of VFA and Ethanol in Fermentation

Altering the conditions of the fermentation for producing alcohols (e.g.lower pH, accumulation of VFA) could further increase the molar ratio ofethanol to VFA produced. Four isolates were incubated in a factorialarrangement with different pH (4, 5, 7) with or without added VFA (2% ofacetate, propionate and butyrate). Each treatment was also tested withthree levels of ethanol at the start (0, 6%, 10% by volume). The addedacids and lowest pH inhibited the production of all products anddecreased microbial growth. These organisms were not well adapted to thehigh VFA concentrations but others could be isolated that are bettersuited to high VFA concentrations by using more VFA in the enrichmentmedia. However, at neutral pH without added VFA, addition of ethanolresulted in much higher ethanol production (2 to 4 fold increase) andnearly complete inhibition (>90%) of VFA production for 3 of the fourisolates tested.

One strategy for ethanol production is to use organisms that toleratecarboxylic acids and alcohols and use these organisms to produce bothalcohols and carboxylic acids. Another strategy is to use organisms andconditions that do not produce carboxylic acids to produce onlyalcohols. Of course, other organisms could be used to produce onlycarboxylic acids. The methods described in this application can provideorganisms and conditions for all three strategies.

Previously, addition of ethanol was shown to inhibit VFA production andto increase ethanol production from cellobiose for several rumenmicroorganisms incubated under conditions to thermodynamically favorethanol production. In addition, ethanol addition was shown to shiftmixed-culture microorganisms in rumen fluid toward ethanol productionwhen incubated under H₂ pressure. Thus, it appears that manymicroorganisms are tolerant to ethanol in the media, and in fact cangrow in the presence of ethanol if other products including gases arecontrolled to make it thermodynamically feasible to produce additionalethanol. In fact, ethanol appears to inhibit VFA production and the highconcentrations of ethanol therefore shifts fermentation toward greaterethanol production.

If the concentrations of gases and other metabolites are not controlled,the higher ethanol concentrations in the media would thermodynamicallylimit additional ethanol production, and together with the inhibition ofVFA production, the organisms would not be able to obtain energy andgrow. Thus, the apparent ethanol intolerance that has been faced byprevious investigators can be overcome by controlling the thermodynamicconditions, especially gas composition and pressures. Controlling thefermentation to maintain some amount of ethanol in the fermentation canvirtually prevent VFA from being produced at all, and therefore wouldfavor further ethanol production, even beyond 10% ethanol by volume ofmedia as was observed in the current study. Further development oforganisms by growing them in media with ethanol would also select forethanol producers and result in degradation of the ability to produceacetate.

Process to Produce Alcohols or Carboxylic Acids

One or more species of isolated microorganism or mixed cultures takenfrom a microbial environment are used in a large-scale process toproduce alkyl alcohol or carboxylic acids from synthesis gases. In thisprocess, certain steps are used to result in optimal ethanol or otheralcohol production.

-   -   1. Optimal conditioning of the media to insure anaerobic        conditions for production.    -   2. Addition of microorganisms that can assimilate gases for        synthesis of the desired product (e.g. alkyl alcohols or acids).    -   3. Maintenance of the optimal temperature for the conversion to        take place.    -   4. Addition of gases to maintain the ratio of synthesis gases        that is calculated to be thermodynamically optimal for the        desired product.    -   5. Maintenance of gas pressures to favor desired products from        the gases.    -   6. Using various means to increase the rate of solubilization of        gases including: mixing, aerating, agitating, vibrating,        increasing surface area, other means or a combination of these        means.    -   7. Removing alkyl alcohols, carboxylic acid, and other products        from the fermenter    -   8. Separating or further treating removed liquids and solids.

The industrial process to produce lower alkyl alcohols or volatile fattyacids uses microorganisms to convert CO₂, CO and H₂ to desired products.These organisms may be aerobic, anaerobic, facultative anaerobic, orstrictly anaerobic. Most alcohols or organic acids are produced underanaerobic conditions in any case. The microbial cultures that are usedcan be mono-cultures or mixed cultures of microorganisms that areselected to predominantly produce a desired product, and which aretolerant to that product.

It may be advantageous to use a pure culture of organisms to preventproduction of undesired products. The pure culture would have a reducedability to produce the undesired products. If using a pure culture,sterile conditions could be used to prevent contamination.Alternatively, some level of contamination may be acceptable if thereactor conditions favor the desired organisms and desired products. Thecultures that can be derived using the methods in this application areideal for this process because these organisms are robust, grow quickly,and produce desired products at a high concentration. These cultureswould readily scale to a large-scale fermentation.

Undefined mixed cultures could also be used and doing so could obviatethe need for sterile conditions. The decision on whether to use a pureculture and sterile conditions depends on the products desired, theextent to which a certain number of specific products is desired, andthe degree to which the conditions favoring a certain product need to bemaintained. For example, with very restrictive conditions (pressures,ratios of gases, pH), certain products can be favored even without usingpure cultures. However, it may be more cost effective to use purecultures and less restrictive conditions. Alternatively, the mixedculture and unrestrictive conditions could provide a mixture of productsthat could be separated and used, and the potentially greater cost ofseparation could be balanced by the lower cost and higher efficiency ofthe fermentation. The best option depends on the market conditions.

Various sources of gases can be used depending on availability andprice. Gases can be derived from heating biomass, fossil fuel (e.g.coal), or other organic product (e.g. plastic) through a process ofgasification. The gases can also be derived from the waste stream of anindustrial or agricultural process. For example, residual gases fromhydrocracking of fossil fuel can be used. Gases can be obtained fromanaerobic digestion with a mixed culture or a pure culture that producesH₂ and CO₂. Hydrogen can be produced from sunlight using algae orplants, or from electrolysis. Carbon dioxide can be obtained fromcombustion or other oxidation process. Steam distillation of methanealso produces gases. For this process, the (CO₂ and H₂) gases do notneed to be separated, which would reduce cost. If the gases only containlow concentrations of CO₂ or H₂, greater total pressures can be used todevelop conditions to make it feasible to synthesize organic compounds.Different sources of gases can be mixed to provide more optimal ratiosof gases. In addition, sources of gases that may contain potentialcontaminants (e.g. sulfide) are also acceptable. The mixed gases may befiltered through a 0.2-micron filter to remove potential microbialcontaminants before feeding the reactors containing the developedmicroorganisms.

In some cases the process may be improved by pressurizing the system toa few atmospheres or more, or by mixing different sources of gases toobtain a desirable ratio, however once the organisms are selected anddeveloped, the exact composition of gases is not as critical. For somemicrobial cultures and for some desired products greater attention topressure and composition of gases is necessary. For example, undefinedcultures would require more precise manipulation of the gas compositionthan pure cultures. Production of alcohol or longer-chain carboxylicacids would require more precise conditions than acetate production.

The composition and pressure of the gases needed would be determinedusing a thermodynamic model that was described as an aspect of thisapplication. The composition and pressure needed to make production of adesired product thermodynamically feasible at a desired concentration iscalculated. Furthermore, the conditions needed to restrict production ofundesired products would also be determined based on the thermodynamicsas described in this application.

Gas is bubbled into the reactors through a gas disperser on a rotatingarm. Alternative methods of gas distribution are also acceptable. Gascan also be solubilized from the headspace by other forms of agitationsuch as vibration or vortexing. The same gas can be re-circulated fromthe headspace, and gas can be replaced as it is used to maintain thedesired pressure.

The reactor vessel is heated to an optimal temperature for growth (e.g.40° C.). Cooler temperatures (e.g. 25° C.) can be used to save energycost but it may slow the fermentation rate. Warmer temperatures (e.g.55° C.) can also be used to increase the rate of fermentation, and tocontribute to decreasing the potential for contaminant organisms. Thetemperature used is suited to the isolate of organism that is used.

The fermentation pH may be regulated by computerized monitoring andadjustment by addition of buffers or bases. The pH may be maintainednear 5 for alcohol production, but some stains grow faster at higher pH(although may make more acids), or lower pH may be used to more stronglyinhibit acid production. Neutral pH favors solubility of CO₂ andbicarbonate (HCO₃ ⁻). Generally, a pH between 4 and 7 has been used inbench-top experiments with organisms exhibiting abilities to makedesired products in this range. However, organisms are known to producealcohols at even lower pH such as pH 2, and a lower pH could be used.

An aqueous broth in the reactor contains simple nutrients (e.g. ammonia,minerals, cofactors). Some microbes can digest and use microbial proteinwhile others synthesize protein from infused gases and ammonia. Bothoptions are possible. Including an organism that digests other microbesat a controlled rate decreases the need to replace nutrients, but alsomay decrease the activity of organisms using the synthesis gases. On theother hand, if the proteolytic activity is minimized the accumulatedmicrobial protein can be removed periodically and the nutrientsreplaced. The microbial protein can be separated and used as ahigh-value animal feed. The protein can be separated by centrifugation,or attachment to another feed product like hay or plant fiber, or byflocculation with an addend.

The organisms that are used for this process tolerate highconcentrations of the desired alcohol or acids or both. The acids aretypically removed by adding a divalent cation such as calcium ions ormagnesium ions, which form a salt with the volatile fatty acids. Thealcohols are commonly removed by distillation. When an organism producesboth alcohol and one or more VFA, the products may be separated by bothmethods. If the organisms begin to show signs of stress due to the overaccumulation of a product, that product may be removed while otherproducts continues to accumulate. The carboxylic acids can beprecipitated even before the fermentation is complete if too much acidaccumulates. If the remaining cation concentration is too high afterprecipitating the VFA, excess cation can be removed by addingcarbonates, which also precipitate with divalent cations. Media can thenbe recycled.

Maintaining microorganisms in the fermenter while removing waste andproducts may be desirable. One way to maintain microorganisms in thedigester is to distill ethanol intermittently using vacuum pressuredistillation, or gas stripping during the fermentation. Alternatively,microbes can be separated from removed liquids with centrifugation orfiltering and returned to the fermenter. However, it may not benecessary to recycle the microorganisms. Liquids can simply be removedand alcohols distilled out, and the liquids recycled to the fermenter.

Processes already being developed using microorganisms to convert CO₂and H₂ or CO and H₂ to acetate, ethanol or other alcohols or carboxylicacids can be adapted using the innovations described in thisapplication. For example, these procedures may be improved by increasingthe pressure of the gases, manipulating the ratios of gases ifwarranted, and using novel organisms isolated using the processdescribed in this application to produce much higher concentrations ofalcohols or carboxylic acids than has been achieved before.

Example Process to Produce Alcohols from Synthesis Gases

For production of alcohols, the optimal ratio of H₂:CO₂ is higher (3:1)than for production of acetic acid (2:1). The production of alcohols isalso more sensitive to the ratio of H₂:CO₂ and total pressure thanproduction of acetic acid. The media pH may be lower than for VFAproduction to favor alcohols over VFA. A pH from 4 to 5, or even lessthan 4 may be used, but a higher pH may also be used to increase therate of production.

Some co-products may be produced, especially VFA when alcohols aredesired, especially if using an undefined culture. These co-products canbe converted to other products or fuels or removed for a subsequentprocess. These products might be catalyzed to ethanol, other alcohols,or alkanes (methane, ethane, propane, butane) using a separate process,which may be a separate fermentation process or a chemical method.Alternatively, the VFA can be converted to H₂ and CO₂ or CH₁, which maybe synthesized to alcohols or used separately. The VFA can also beconverted to alkyl alcohols using low-pH conditions and gasconcentrations that simultaneously favor alcohol degradation andsynthesis of alcohols.

One unique aspect of this approach is the organisms produce highconcentrations of ethanol, such as greater than 6%, and preferablygreater than 8% or even more preferably greater than 10% ethanol byvolume. With further improvement of organisms using the processdescribed in this invention, as much as 20% alcohol may be produced.Another aspect is the possibility of producing longer chain alcoholslike propanol and butanol.

Adjusting the gases to a ratio of H₂ to CO₂ of 3:1 increases thethermodynamically feasible concentration of ethanol that can be producedfrom the gases, and increases the concentration of ethanol that isobtained. Another way to increase the thermodynamically feasibleconcentration of alcohol produced is to increase the pressure aboveatmospheric pressure. For example, even if very low concentrations of H₂and CO₂ result from the waste stream of a process, these lowconcentrations can be captured by providing some additional gas (CO₂ orH₂) to provide a ratio of H₂ to CO₂ that is readily used for alcoholproduction (e.g. 3 to 1). In addition the total pressure can beincreased, which increases the partial pressures, and makes it feasibleto produce greater concentrations of the alcohol.

Example of Producing Carboxylic Acids from Gases

Similar procedures to those used for alcohol production can be used toproduce acids, such as VFA, rather than alcohols but the ratios of gaseswould be adjusted to mainly produce the desired acids. Because acids area common product of mixed culture fermentations, if a mixture of gasesis desired, aseptic conditions and defined microbial cultures would notbe needed. The profile a gases can be controlled by adjusting thethermodynamics. Longer-chain carboxylic acids are favored by increasingtotal gas pressures, increasing the ratio of H₂:CO₂ or H₂:CO anddecreasing pH. However, defined cultures can be used to obtain a singleor limited number of desired acids, such as acetic acid, propionic acid,or butyric acid. The acids can be used subsequently for alcoholproduction in a different process, which may be a chemical processrather than a fermentation process. For example, acetate may beconverted to ethanol, propionate may be converted to a propanol, orbutyrate or acetate may be converted to butanol. Any of these acids maybe converted to alkanes, including longer chain alkanes, in a subsequentprocess. Or they may be used to construct bio-polymers or they may beseparated and sold as chemicals. Free fatty acids can be precipitatedwith divalent cations (e.g. Ca²⁺, Mg²⁺) to form salts such as calciumacetate or magnesium butyrate. If VFA are produced as byproducts ofalcohol production, both may be removed and used. For example, someorganisms produce some acetate and ethanol and the acetate can beremoved by adding CaOH and causing it to precipitate. If theconcentration of calcium becomes too high, it too can be precipitated byadding bicarbonate to form calcium carbonate.

Example of Producing Animal Feeds from CO₂, CO and H₂

Mixed rumen microbes taken directly from the rumen of a cow wereincubated in vitro while perfusing different mixtures of CO₂ and H₂, andthe total microbial protein and VFA concentrations increasedsubstantially compared to when gases were not perfused. Interestingly,the profile of VFA changed slightly, and all VFA measured increased(acetate, propionate, butyrate, valerate, iso-valerate, iso-butyrate).Cattle obtain much of their protein from the high-quality microbialprotein synthesized in the rumen during degradation of substrate. Theyobtain most of their energy from the VFA produced by thesemicroorganisms. This example shows that synthesis gases can be used toproduce microbial protein and VFA, both of which can be used for animalfeeds. The microbial protein could be used for cattle or most otheranimals. Many processes produce one or more of the synthesis gases thatcan be converted to other products including animal feeds.Interestingly, in this example pure cultures were not needed. Undefinedmixed cultures produce many products but they don't need to be sterileor aseptic and they do not have requirements for as many micronutrientslike vitamins because some of the microorganisms can produce thesevitamins.

An undefined mixed culture or a pure culture of microorganisms would beprovided with needed micronutrients for growth including minerals andvitamins, and buffers or the regulated addition of bases would be usedto maintain neutral pH. A source of nitrogen and sulfur would beincluded. The fermenter may need a source of amino acids or protein insome cases, or it may need ammonia or urea in other cases, depending onthe microbial culture. Gases will be perfused into the culture possiblyusing a bubble disperser or other method to increase solubility,pressures may be applied, gases may be re-circulated, and ratios ofgases may be adjusted to increase the rate of production or to selectfor desired products. The products can be fed to animals directly, orafter drying or mixing with other feed, or they may be separated andused for separate processes or types of animals. Even when the VFA oralcohols are used in one process, the microbial protein can be fed toanimals.

Thermodynamics for Synthesis of Alcohols or Acids from CO₂, CO and H₂.

The process used as an aspect of this invention advances thermodynamicanalysis in many ways. Firstly, multiple thermodynamic equations weresolved simultaneously to enable prediction of which pathway branches areavailable, and to integrate the effects of each reaction on each other.Secondly, thermodynamics was applied to the fitness of organisms thatcarry out a given reaction relative to other organisms. If the ΔG forCO₂ and H₂ to acetate is much more negative than for CO₂ and H₂ toethanol for a certain set of concentrations and pressures, organismsthat make acetate will grow faster than those that make ethanol. Toencourage ethanol production within an organism, or to encourageorganisms that make ethanol to out compete with acetic acid producers,the metabolite concentrations must be manipulated to favor ethanolproducers and production of ethanol. Under these conditions, ethanolproducers can be used, enriched, selected, mutated and manipulated. Thesame procedure is used for any desired product and will also bedemonstrated for other alcohols and for different carboxylic acids.

Conditions that Affect the ΔG for Synthesis Reactions

In a natural microbial ecosystem, there are many different reactionsthat can use H₂ and CO₂ to produce organic compounds when theconcentrations of H₂ and CO₂ are high enough. When the concentrations ofthe gases are low, the organic compounds are degraded to H₂ and CO₂instead. Which direction the pathways flow depends on the change in freeenergy of the reaction. Microorganisms can only grow if they capturesome of the benefit of the free energy change (ΔG). For example,typically about 44 kJ of energy in ΔG is used to convert a mole of ADPto ATP.

The ΔG for different concentrations of reactants and products andtemperatures can be calculated as described previously (ΔG=ΔG°+ln{[products]/[reactants]}). Where this value is a negative number, thereaction can proceed spontaneously in the forward direction. Where it isa positive number, the reaction cannot proceed without the input ofenergy or it may proceed in the reverse direction. The magnitude of theabsolute value of the ΔG indicates how much energy may be paired withthe reaction. For example, a ΔG less than (more negative than) −44 kJindicates the potential for production of one mole of ATP.

The calculated ΔG values for three reactions important to fermentationand synthesis from gases are shown in FIG. 1. In this example, aconstant total pressure of 1 atm was maintained, but the ratio of H₂ toCO₂ increased. At the left end of the diagram, typical conditions in ananaerobic fermenter or the rumen of the cow are shown. Note that the ΔGfor methane synthesis from 4H₂ and 1 CO₂ is about −50 kJ/mol of methaneproduced. Therefore, it would be possible to produce about 1 ATP permole of methane. The corresponding ΔG for acetate or ethanol synthesisfrom H₂ and CO₂ are positive. While the ΔG for acetate synthesis is near0, that for ethanol synthesis is strongly positive (+75 kJ/mol). Underthese conditions, ethanol can be converted to acetate or might bedegraded to CO₂ and H₂. As the H₂ to CO₂ ratio increases, the ΔG formethane continues to decrease and then begins to slowly increase. Theminimum value occurs at a ratio of 4:1H₂ to CO₂ on a molar basis orvolume basis. At the minimum, nearly 3 ATP could be generated per mol ofmethane rather than one. This explains why methanogens grow faster underthese conditions. In addition, as the ratio of H₂ to CO₂ increases, italso becomes thermodynamically feasible to produce acetate from CO₂ andH₂. The minimum value occurs at a ratio of 2:1H₂ to CO₂, and it ispossible to produce more than one ATP per mole at this ratio. The ΔG forethanol synthesis decreases as well until it reaches a minimum at aratio of 3:1H₂ to CO₂. Wherein the ΔG for ethanol synthesis initiallydecreases faster than for acetate synthesis, and the ΔG for both acetateand ethanol decline faster than for methane, the curves converge as theratio increases toward the minimum. Thus, near the minimum, it becomespossible to make all of the products. After reaching the minimum ΔG, thecurves for acetate and ethanol increase faster than for methane, makingit more favorable to produce methane than the other two as the H₂ to CO₂ratio increases further.

This graph explains many decades of research results conducted withanaerobic systems. It is clear from this graph why methane is thefavored product under typical fermentation conditions and why methane isespecially favored at low or high ratio of H₂ to CO₂. If thefermentation is designed to degrade acetate to methane and CO₂, either alow or a high ratio of H₂ to CO₂ is required. Furthermore, acetate andethanol are only produced when there is both H₂ and CO₂ present. If thepH is decreased, the curve for acetate would increase to approach thatof ethanol. If the pressure is increased, all of the curves woulddecrease but the ethanol curve would decrease the most, the acetatecurve would decrease the second most, and the methane curve woulddecrease the least. Thus, decreasing pH or increasing total gaspressures are ways to favor ethanol or both ethanol and acetate overmethane.

The ΔG for production of ethanol, propanol, and butanol are shown inFIG. 2. Initially, production of alcohol is not feasible under theconditions shown. As the H₂ to CO₂ ratio increases, first butanol, thenpropanol, and finally ethanol cross the line Y═o representingthermodynamic feasibility. At H₂ to CO₂ ratio of 3:1, each ΔG isminimized with the longer alcohols more favored than the shorter ones.Thus, conditions of a ratio of 3:1 favor any alcohol production withenough energy left over for ATP production. The same conditions forcarboxylic acid production are shown in FIG. 3. In this case, acetate isthe first to become thermodynamically feasible, but as the H₂ to CO₂ratio increases longer-chain carboxylic acids also become feasible, withthe longer acids more favored than the shorter ones.

These data show how to establish conditions for synthesis of alcohols(including longer-chain alcohols) or longer-chain carboxylic acids fromH₂ and CO₂. Wherein many different potential reaction pathways competefor the H₂ and CO₂ these conditions need to be understood to select fororganisms to make certain products or to establish conditions in thedigester to make it feasible, and even favorable, for the desiredproducts. Note that by “feasible” we mean the ΔG is negative and it ispossible to make the product. By “favorable” we mean the ΔG is negativefor the desired product and ΔG may be positive (or at least lessnegative) for a major competing reaction. Thus, the desired product willbe produced to a greater extent than other products.

Example Calculations from Thermodynamic Model

Although a model encoded with software provides much greater flexibilityfor exploring the potential to control a fermentation system, examplecalculations are presented in Table 5. The accuracy of the model dependson how well the fermentation is defined in terms of what reactions areavailable in the system (what enzymes are present), the amount of ATPgenerated per reaction, the free energy of formation of each reactantand product, and the stoichiometry of the reactions. The free energy offormation is easily obtained from textbooks. The stoichiometry ofreactions is generally clear for synthesis reactions from CO₂, CO andH₂. For example, Two CO₂ and 6 H₂ are needed to balance production of 1ethanol (C₂H₅OH) and 3 H₂O. The number of ATP per reaction also variesand the fermentation system could enable more than one option for ATPproduction. Generally, degradation or synthesis of ethanol or aceticacid from or to gases only yields a fraction of an ATP (e.g. 0.2 ATP).Despite these uncertainties, the relative differences in the way eachmetabolite is affected by these conditions are clearly established.

Equilibrium concentrations were calculated to represent addition orremoval of H₂ or increased total pressure of the fermentation (Table 5).These values may vary from observed values because of uncertainties ofefficiencies, however the trends in concentrations relative to othermetabolite concentrations depend on known stoichiometry, and thus aremore certain. The predictions show, for example, the impact of adding orremoving H₂ to establish new H₂ concentrations on methane production andacetate degradation or production. For example, removing H₂, asdescribed in U.S. patents filed by the inventors, U.S. Ser. No.12/000,856 and 60/871,441, is represented in the far left column andshows the greater degradation of acetic acid and capture of more energyas removed H₂ rather than CH₄ or other VFA. Increasing H₂ increases CH₄production. The amount (moles) of H₂ removed or added can also becalculated from stoichiometry using the model. Although the effects onCH₄ production are opposite for increasing or decreasing H₂, acetic aciddegradation increases for both effects. Thus, manipulating the ratio ofH₂ to CO₂ is a means to shift fermentation toward degradation of acetateto methane or the opposite effect.

The far right columns show the effect of increasing the total gaspressures. The higher total pressure does not affect the pressure of H₂,but H₂ becomes a lower percentage of the total gas. This of course isthe opposite effect of decreasing pressure using application of vacuum(U.S. Ser. No. 12/000,856 and 60/871,441). The equilibriumconcentrations of acetic acid increase with higher total pressure. Thus,increasing pressure shifts the fermentation toward greater acetic acidproduction (either from CO₂ and H₂, or by decreasing degradation) ratherthan production of methane and H₂. The thermodynamic model readilydemonstrates the effects of various manipulations. Furthermore, thesemanipulations can be used to select for a type of microbial activity, toisolate microorganisms with that activity, and to direct evolution. Forexample, gas pressures needed to select for bacteria that produceacetate from CO₂ and H₂ can be determined as shown.

TABLE 5 Selected equilibrium pressures and concentrations when partialpressures of gases are manipulated. Item Total pressure = 1 atm Totalpressure = 2 atm CO₂, atm 0.95 0.75 0.50 0.25 0.05 1.90 1.0 0.10 CH₄,atm 0.05 0.25 0.50 0.75  .95 0.10 1.0 1.90 H₂, atm 1.0 × 10⁻⁴  1.6 ×10⁻⁴  2.2 × 10⁻⁴  2.8 × 10⁻⁴  4.5 × 10⁻⁴  1.0 × 10⁻⁴  2.2 × 10⁻⁴  3.8 ×10⁻⁴  Ac 2 × 10⁻³ 8 × 10⁻³ 1 × 10⁻² 8 × 10⁻³ 2 × 10⁻³ 8 × 10⁻³ 4 × 10⁻²4 × 10⁻³ degradation if greater: Ac synthesis 2 × 10⁻⁶ 9 × 10⁻⁶ 1 × 10⁻⁵9 × 10⁻⁶ 2 × 10⁻⁶ 9 × 10⁻⁶ 5 × 10⁻⁵ 4 × 10⁻⁶ if less:Model assumes pH=6.5, temperature=39° C. Ac=acetate, at 50 mmol/L.Assumes production of 0.2 mol interconversion of ATP with ADP per molacetate synthesis or degradation.

The results in Table 5 demonstrate a means to control a mixed culturefermentation containing methanogens to produce more acetic acid and lessmethane by increasing pressure. Alternatively, acetate degradation canbe increased by decreasing pressure or by increasing or decreasing theratio of H₂ to CO₂. Decreasing CO₂ or increasing H₂ can shift thefermentation toward making more methane and degrading the acid.

The inventors isolated and used microorganisms that synthesize alkylalcohols, such as ethanol, from CO₂ and H₂ or CO and H₂. The inventorsdemonstrated that not only is de novo synthesis (from CO₂ and H₂) apossibility, but also that degradation of acetic acid and ethanol can becarried out by the same microorganisms and the same enzymes. Whetherethanol or acetic acid are created from CO₂ and H₂ or degraded to CO₂and H₂ depends on thermodynamics, and concentrations of reactants orproducts. If the partial pressure of gases is high relative to theconcentration of ethanol or acetic acid, the equilibrium is shiftedtoward synthesis of ethanol or acetic acid rather than degradation.

Ways to Shift Fermentation Regarding Synthesis or Degradation of Acetateand Ethanol

There are several ways in which the thermodynamic analysis shows it ispossible to shift metabolism toward synthesis of a desired alcohol oracid. The first way is to change the pressure of all gases, for exampleto increase total gas pressures, so that the partial pressure of CO₂ andH₂ are affected. Increasing the partial pressure of all gases this wayincreases the concentration to which alcohols or acids can besynthesized from gases. Decreasing the partial pressure of all productgases increases the degradation of acetic acid or ethanol. The secondway to increase synthesis of alcohols or acids is to adjust the ratio ofsynthesis gases to a ratio that favors a certain product, the higherratio favoring alcohols over acids and longer-chain length acids oralcohols over the shorter. In addition, low pH also favors alcohols overacids.

Calculations of ΔG or equilibrium calculations can be used to quantifythe effect of the ratio of H₂ to CO₂, the total pressure, and pH. Thesecond law of thermodynamics enables calculation of which direction apathway flows and whether energy is required or can be captured bycarrying out the reaction. The relationship is determined by the signand magnitude of the change in free energy (ΔG) from the equation:

ΔG=ΔG°+RT Ln {[Products]/[Reactants]}

where [Products] or [Reactants] represents the multiplicative product ofthe concentrations or partial pressures of solutes or gases and ΔG° isconstant for each reaction based on the products and reactants. When theΔG is negative, the reaction can proceed and if it is negative enough,ATP can be generated.

Another way to represent the direction or feasibility of reactions is tocalculate the equilibrium concentration of a product. Additional productcan be feasibly accumulated if it the concentration is less then theequilibrium concentration, or the product will be degraded if theconcentration is less than the equilibrium concentration. Theequilibrium concentration is calculated by setting the ΔG to 0 andsolving the equation for the concentration of products. The ΔG mayinclude the ΔG for ATP generation if it is known or presumed to supportmicrobial growth. This approach was used to show the effect of pressure,ratio of H₂ to CO₂ and pH on the concentration of alcohols andcarboxylic acids. For the following reactions:

6H₂+2CO₂←→ethanol+3H₂O,ΔG°=−104.1 kJ/mol

4H₂+2CO₂←→acetic acid+4H₂O,ΔG°=−71.6 kJ/mol

The feasible concentration of acetate equalse^((*ΔG*-ΔGATP)/RT)×[H₂O]²[H⁺]×[H₂]⁴[CO₂]². The ([H⁺] is equal to −log₁₀pH, and [H₂O] was assumed to be 50 mol/L. The result depends on theamount of ATP produced. In this example, free energy for ATP was assumedto be 44 kJ/mol of acetate produced. The feasible concentration ofethanol equals e^((ΔG-ΔGATP)/RT)×[H₂O]³×[H₂] [CO₂]². Feasible (orequilibrium) ethanol concentration does not depend on pH. ATP was againassumed to be 44 kJ/mol of ethanol produced. Equilibrium concentrationsof other alcohols and acids were determined in a similar manner solvingfor the concentration of each carboxylic acid or alcohol under differentpressures or ratios of H₂ and CO₂.

As longer VFA and alcohols include additional synthesis steps, thenumber of ATP (and therefore kJ/mol captured) was assumed to be greaterby 1 ATP (44 kJ/mol) for each elongation step. For example, synthesis ofpropionate or propanol was assumed to capture 2 ATP (88 kJ/mol), andsynthesis of butyrate or butanol was assumed to capture 3 ATP (132kJ/mol). These values reflect the steps known for ATP capture in thepathways for interconversion.

The equilibrium concentration of ethanol or acetic acid synthesisrelative to degradation at a constant total pressure is shown in FIG. 4.If the total gas pressure is maintained at 1 atm, as the H₂ pressureincreases from 0 atm to 1 atm, the CO₂ partial pressure will decreasefrom 1 atm to 0 atm. The estimated peak concentrations of acetate orethanol would occur when the ratio of H₂:CO₂ is 2:1 for acetic acid, and3:1 for ethanol (FIG. 4). Although temperature and total pressureaffects the extent to which ethanol or acetic acid can be concentrated,the ratios of gases to maximize the concentrations of ethanol or aceticacid under any given set of conditions is constant. One way to increaseethanol synthesis from gases, is therefore to add or retain either CO₂or H₂, or remove or utilize CO₂ or H₂ to leave gases in thecorresponding ratio for maximal synthesis. A way to increase degradationof ethanol or acetic acid is to manipulate CO₂ or H₂ away from theratios for maximal synthesis by either increasing or decreasing CO₂ orH₂.

Thermodynamics also shows that increasing total gas pressure is a meansto increase both CO₂ and H₂ partial pressures, thereby increasingsynthesis over degradation of ethanol or acetic acid. Concentrations ofboth ethanol and acetic acid that can be synthesized increaseexponentially as the total gas pressures increase at a constant ratio ofH₂ to CO₂ (FIGS. 5 and 6). However, exponent is 8 for ethanol and 6 foracetic acid corresponding to the stoichiometry of ethanol and aceticacid synthesis, which requires 8 or 6 moles of gas respectively toproduce ethanol or acetic acid. Thus, the effect of pressure isexponentially greater for ethanol synthesis than for acetic acidsynthesis (FIG. 7), where the exponent is 2. In other words, increasingtotal gas pressure when the gases are comprised of a similarconcentration of CO₂ and H₂, shifts metabolism toward synthesis of bothethanol and acetic acid, but also shifts metabolism toward ethanolrelative to acetic acid.

The feasible concentration predicts only the acetate form, not theconjugate acid. But at neutral pH most of the acetic acid and acetate isin the acetate form. At near neutral pH, the thermodynamically feasibleconcentrations are high for carboxylic acids. For example, assuming 1ATP produced per mole of acetate produced, pH 6.5, and 1 atm of H₂ andCO₂ in the ratio for maximal acetate synthesis, peak acetate yield atequilibrium could be 442 M when solving for the equilibriumconcentration using the ΔG equation. This is far beyond the number ofmoles that could fit in the space indicating that there would be nothermodynamic limit to acetate production under these conditions. If thepH in the above example is decreased to 4, the acetate production wouldbe limited to about 1.4 M (and a slightly greater amount of the aceticacid form would also be produced), but the ethanol concentration wouldnot be affected by pH.

These discoveries can be applied to increase the efficiency of ethanolproduction from CO₂ and H₂, by using high total pressures to increasethe ethanol concentration that can be derived from gases. It can also beused to enrich or select for microorganisms that synthesize ethanolrather than acetic acid, or to control microbial cultures that haveaccess to both pathways. The inventors found empirically that whenincubating with two atmospheres H₂ pressure, organisms were isolatedthat decreased total gas pressure while they fermented cellulosicsubstrate. These organisms synthesized additional ethanol from the addedH₂ and the CO₂ released from degradation of cellulosic biomass toethanol. The inventors also isolated organisms under conditions favoringethanol production (4 atmospheres, ratio of H₂:CO₂ of 3, and thoseorganisms produced a higher molar concentration of ethanol than acetate.In particular, isolates obtained after enrichment in pH 5 media producedmore ethanol than isolates enriched on pH 7 media.

In an industrial process, most of the gases will be able to be convertedto ethanol, rather than acetate, if conditions do not favor acetateproduction or if the microbes that make ethanol do not make acetate.Both of these are possibilities. If acetate is allowed to build upinitially, it inhibits further acetate production as ethanol isproduced. The acetate can be maintained in the fermenter as ethanol isremoved (distilled out), or both can be removed and used separately. Onthe other hand, growing organisms under conditions that disfavor acetateproduction selects against acetate producers and eventually providesorganisms with impaired ability to produce acetate. In studies conductedby the inventors, some isolated organisms produced a much higher molarratio of ethanol to acetate than others even when isolated under thesame conditions. This suggests the ability for the microbes to adapt toproducing ethanol making it less critical to always maintain thoseconditions to continue producing ethanol.

Thermodynamics for Synthesis from H₂ and CO

The same principles can be applied to mixtures of H₂ and carbon monoxide(CO). Similar calculations based on stoichiometry can be used toquantify that the ratio of H₂ to CO for maximal synthesis is 2:1 forethanol or 1:1 for acetic acid. The effect of increasing pressure is thesame as for synthesis of ethanol or acetic acid from H₂ and CO₂. Thus,adjusting gas formulations and increasing pressures would also shiftmetabolism toward greater ethanol synthesis or acetic acid synthesiswhen using CO and H₂ as well, or when selecting for microorganisms thatcan use these gases. The inventors discovered that organisms selected toproduce products from CO₂ and H₂ produced a similar profile of productsfrom CO and H₂.

Thermodynamics for Synthesis of Other Alcohols

These principles were also applied to determine the optimal ratios forbutanol or butyrate synthesis (FIG. 8). The optimal ratio of H₂ to CO₂is 3:1 for butanol production or 5:2 for butyrate production. Increasingtotal pressure at a constant ratio of gases increases butanol andbutyrate equilibrium concentration, and butanol concentration relativeto butyrate (FIG. 9). The optimal ratio of H₂ to CO for butanolsynthesis was 2:1, and the optimal ratio of H₂ to CO was 3:2 forbutyrate synthesis. The effect of pressure was similar for H₂ and CO asfor H₂ and CO₂.

Controlling the ratio and total pressure of H₂ and CO₂ favors long-chainalcohols and long-chain acids over acetate. For example, at twice thepressure (2 atm vs. 1 atm) the equilibrium concentration of acetateincreases 70 fold, but the equilibrium concentration of ethanolincreases more than 250 fold. These values were calculated from the peakresults of FIGS. 4 and 5, by comparing the peak reactant concentrationsat the higher pressure to those at the lower pressure. At 2 atm pressureof total gases, the equilibrium concentration of butyrate would increase17,000 fold over 1 atm (compared to 70 fold for acetate), and theequilibrium concentration of butanol would increase 80,000 fold over theconcentration at 1 atm. Thus, it is apparent that the effect of pressureand ratio of H₂ to CO₂ is much more important for alcohols over thecorresponding acids, and for longer chain alcohols or acids over theshorter chain length.

These principles also apply to synthesis of propanol and propionate fromgases. The optimal ratio of H₂ to CO₂ is 3:1 for propanol synthesis or7:3 for propionate synthesis (FIG. 10). Increasing total pressure at aconstant ratio of gases increases concentration where propanol andpropionate synthesis occur and increases concentration where degradationoccurs, and increases possible propanol concentration relative topropionate. The optimal ratio of H₂ to CO for propanol synthesis was2:1, and the optimal ratio of H₂ to CO was 4:3 for butyrate production.The effect of pressure was similar for H₂ and CO as for H₂ and CO₂.

In every case, increasing gas pressures shifted metabolism toward highersynthesis concentrations, and away from degradation, and toward greateralcohol compared to the analogous volatile fatty acid concentration. Theactual calculated concentrations are the optimal mixtures of gases foreach of these synthesis reactions, and one aspect of the invention isthe use of thermodynamics and stoichiometry to calculate optimal ratiosof gases for synthesis of desired products. Another aspect is theapplication of total gas pressures, such as greater than 1 atm or more,to increase the concentrations that alcohols or acids when produced fromgases. This concept can be applied to any fermentation reaction in whicha greater number of moles of reactant gases are used to produce a lowernumber of moles of product gases, and the reaction, like manyfermentation reactions, is near equilibrium.

Providing partial pressures of gases that make it thermodynamicallyfeasible to produce a high concentration of carboxylic acids withgreater than 2 carbons (e.g. C₃ to C₂₀) or to produce a highconcentration of alcohols, is not likely to be achieved reliably withoutunderstanding and calculating the thermodynamic constraints. Simplyadding H₂ or CO₂ to the reactor would shift the fermentation toward oneor the other ends of the diagrams in FIGS. 4 to 5 and 8 to 10, andresult in degradation of the alcohols or longer-chain acids at leastsome of the time. Applying higher pressure without providing the correctratio of gases would also be futile. Only through using thethermodynamic analysis and with use of microorganisms that are selectedthrough this process, is it possible to consistently produce the desiredproducts at a high concentration. For this reason, microbes have notbeen previously isolated that are known to produce the significantquantities of longer chain acids or alcohols from CO₂, CO and H₂ andprevious investigators have not been able to produce high concentrationsof either acids or alcohols from the gases.

When a ratio of H₂ to CO₂ is perfused into the incubation that is notoptimal for the desired end product, one of two events must occur: 1)the metabolism will shift in order to use the excess gas, or 2) theaccumulation of the desired product will stop because the gas mixturewill shift toward a greater concentration of the excess gas as thelimiting gas is used up. The first case occurs when an alternativepathway is available, and it will result in production of undesiredproduct rather than the desired product. The second case would beespecially limiting for microbial cultures with limited options to shiftto different end products. These organisms would appear to be intolerantto high concentrations of the product they produce. In fact, they wouldbe limited thermodynamically at one end or the other of the previousgraphs 4 to 5 and 8 to 10 (i.e. low CO₂ or low H₂). One means to selectfor organisms to produce a desired product from synthesis gases is tomaintain the optimal ratio of gases for the desired product.

Furthermore, the thermodynamics described above led to a betterunderstanding of what type of microbial activity is needed to producealcohols from CO₂, CO and H₂. Wherein, production of high concentrationsof alcohols is promoted by moderately low pH (e.g. pH 4 to 5) and highpressures of H₂, organisms that can tolerate these conditions wereisolated. Using the partial pressures of H₂ and CO₂ to favor alcoholproduction or longer chain carboxylic acid production resulted inisolation and use of microbes that could produce ethanol, 1-propanol, or1-butanol in the presence of a high concentration of these alcohols. Forexample, microbes produced ethanol from synthesis gases in the presenceof more than 10% ethanol concentration.

Thermodynamics for Synthesis of Carboxylic Acids

Just as the conditions to favor synthesis of alcohols of differentlength were established through the previous analysis, the approach canalso be applied to produce volatile fatty acids (e.g. C₂ to C₅), or evenlonger-chain carboxylic acids. The ΔG values shown in FIG. 3 demonstratethe potential for producing carboxylic acids of increasing length byusing a ratio of H₂ to CO₂ to make production of desired carboxylicacids feasible or more favorable than competing products. The minimal ΔGfor acetate production from H₂ and CO₂ occurs at 2:1H₂ to CO₂ ratio, butthe minimal ΔG for longer carboxylic acids occurs at a slightly higherratio (up to 3:1). The thermodynamics of longer carboxylic acids is alsofavored by increasing total pressure and lower pH favors longer chainacids over the shorter ones. These are precisely the conditions in whichorganisms that mainly produce longer chain acids like butyrate andiso-valerate were isolated. The inventors measured free carboxylic acidsas long as caproic acid (C₆) from mixed rumen fluid so these longeracids can be produced and excreted from the organisms. Typically, thesecarboxylic acids would not be produced in a natural fermentation, butthey can be produced if the ratio of H₂ and CO₂ is increased abovenatural conditions.

It is clear from FIG. 1, that the conditions of pressure and gascomposition for acetate production are not as stringent as for longerchain carboxylic acids or alcohols. However, it is helpful to maintainadequate CO₂ and H₂ pressure and pH>4 or more preferably greater than 5.Producing acetic acid as a feedstock for another process or as a productit self is one of the easiest organic compounds to synthesize from gasesunder typical fermentation conditions. In fact, it may be useful torecover gases that have low CO₂ or H₂ composition by converting thegases into acetate. For example, gases with too little H₂ to be worthyof separation in a cost efficient manner, or gases from fermentation orcombustion could be used. The waste gases may be pressurized to make itpossible to use skewed ratios or low percentages of H₂ or CO₂. It may behelpful to maintain to maintain neutral pH for the process, but someorganisms are acid tolerant. Alternatively, this analysis shows how tolimit acetate production thermodynamically by decreasing pH.

The thermodynamic analysis further shows that greater care needs to betaken to establish conditions for alcohol or longer-chain carboxylicacid production. In fact, it is unlikely that very high concentrationsof ethanol can be produced without understanding the exact conditions ofpressure and gas composition that are needed. In addition, one needs tounderstand the exact conditions to disfavor acetate production (e.g. lowpH) to limit its production. Finally, microbes to produce longeralcohols require even more exact conditions, including pressurizationabove 1 atm and potentially above 2 or 3 atm and a ratio of H₂ to CO₂(e.g. 3) or H₂ to CO (e.g. 2) that maximizes production of thelonger-chain acids or alcohols. When these conditions were applied, theinventors isolated microbes that made significant concentrations ofthese alcohols (ethanol, 1-propanol, 1-butanol), and various lengths ofcarboxylic acids (C₂ to C₅) that were enriched for, selected, and usedto make desired products.

Certain Embodiments of the Invention

The process described herein comprises using multiple simultaneousequations to calculate ΔG for all possible reactions in a fermentationsystem for production of organic compounds from H₂, CO₂, and/or CO. Themathematical model differs from models used previously in that multiplesimultaneous equations are used and solved to identify the optimalconditions to isolate and develop organisms and conduct thefermentation. By using simultaneous equations, it is possible todetermine conditions that make desired pathways thermodynamicallyfavorable compared to competing pathways, and therefore to shift thefermentation in the desired direction.

The models indicate the composition of gases (e.g. CO₂, CO and H₂)necessary to make production of high concentrations of certain alcoholsor acids thermodynamically feasible or favorable, and these conditionsare used in conjunction with other information to isolate, develop anduse microorganisms for optimal production of various alkyl alcohols andcarboxylic acids. The models and empirical results also show theadvantages of pressurizing the fermentation above 1 atmosphere forproducing a higher concentration of the desired product. Many otherconditions such as temperature, pH, and concentrations of metabolitesfurther improve the fermentation toward desired results. All of thesemanipulations are aspects of the process, however, the process alsoembodies more than each of these individual manipulations.

The process involves manipulation of the gases and other products (e.g.alcohol concentration, carboxylic acid concentration) and temperature inconcert with each other to direct the fermentation to produce desiredproducts. Although many different sets of conditions can be used, thisapplication shows how to evaluate and optimize appropriate sets ofconditions for a certain product concentrations. For example, the modelcan be used to develop conditions that make it thermodynamicallyfavorable to produce certain concentrations of desired products. Thus,rather than prescribe a certain ratio of gases or a certain pressure ofthe fermentation, the process described in this application enables theuser to determine the optimal ratio of gases, pressure and other factorsthat are needed together to obtain the desired results. For example, themodel can determine if it is more cost effective to pressurize thesystem than to adjust the ratio of gases.

No previous investigator has been able to make high concentrations ofalcohols or carboxylic acids, or to shift fermentation toward certainproducts, because they have not used all of the conditions required tomake it thermodynamically feasible, and certainly not thermodynamicallyfavorable, to produce those products. Although it is sometimes possibleto occasionally achieve the appropriate conditions by coincidence toproduce some products, the thermodynamic model makes it possible toconsistently produce the product in high concentration and to maintainthe organisms that produce it. Without understanding thermodynamicaspects of fermentation, it is impossible to consistently producealcohols or longer chain carboxylic acids from synthesis gases.

Existing processes using current microorganisms may be improved by usingthe defined process conditions from this application to make greaterconcentrations of alkyl alcohols or carboxylic acids, but it would beespecially advantageous to use the process conditions described in thisapplication with microorganisms that are isolated or developed using theprocess described in this application. Conditions were described andused to enrich for microorganisms that produce the desired alcohols orcarboxylic acids from H₂, CO₂, and CO. Conditions were established toisolate microbial isolates on the basis of the products they producebecause the conditions are such that organisms that produce certainproducts are favored. In addition, the model is used to establishconditions that can be used to select from mutants that produce more ofthe desired products, or a higher rate of production, or less of anundesired product. In this way, the conditions improve microorganismsmaking them better able to produce a desired product. The microorganismsthat were isolated and developed as an aspect of this application canproduce high concentrations of ethanol, such as more than 10% ethanol byvolume, from CO₂ and H₂ or CO and H₂. Isolates were also isolated thatwere tolerant to 6% propanol and 6% butanol and that produced 1-propanoland 1-butanol. Some of the isolates were aerobic or aerotolerant,offering certain advantages of handling.

Although many variations are possible to produce products from varioussources of gases (CO₂, CO, and H₂), key features of this inventioninclude the simplifications of the procedure commonly used, ability touse lower concentrations of gases, ability to obtain greaterconcentrations of the desired products at higher rates, and ability toobtain some products that have not been obtained previously fromsynthesis gases.

There are several novel aspects of the process described in thisapplication including but not limited to those aspects summarized below.Several microorganisms were isolated that convert hydrogen gas andcarbon dioxide gas or carbon monoxide gas and hydrogen gas to ethanol inaqueous media wherein ethanol concentration exceeds 4% by volume. Thesemicrobes were found to be tolerant to more than 10% ethanol by volumeand to grow and produce additional ethanol under these conditions. Themicrobes also tolerate more than 1% acetate, and preferably more than 2to 3% acetate in the fermentation media. In some cases multiple isolatesof organisms can be used together or pure cultures of individualisolates can be used. In some cases, the amount of ethanol produced wasgreater than the amount of acetic acid produced on a molar basis. Manyof the isolates were tolerant to oxygen, and at least one isolate wasidentified as a member of the genus, Enterococcus by 16S rRNA sequence.It had greater than 99% homology with Enterococcus avium. Many of theisolated microorganisms produced ethanol and grew at a pH less than 5,and sometimes less than pH 4.

A method was described for producing ethanol from hydrogen gas andcarbon dioxide gas or from hydrogen gas and carbon monoxide gas or acombination of both. In this method, the ethanol concentration couldexceed 4% by volume in aqueous media, but could also exceed 7% or 10% byvolume. The method may comprise pressurizing the reaction vessel togreater than 1 atm, or even greater than 2 atm total gas pressure. Inthe method, the ratio of hydrogen to carbon dioxide in volume may begreater than 2 to 1 or more preferably greater than 3 to 1. The ratio ofhydrogen to carbon monoxide would be greater than 1 to 1 or morepreferably greater than 2 to 1. Under these conditions of pressure andratio of gases, and other conditions of temperature and ethanolconcentration, the ΔG for ethanol production is less than 0 even as theethanol concentration increases. The pH in the fermentation may be lessthan 7 or more preferably less than 5, and it may even be less than 4.Under these conditions of pressure and ratio of gases, and conditions ofpH and ethanol and acetate concentration, the ΔG for ethanol productionmay be more negative than the ΔG for acetate production, thus making itmore favorable to produce ethanol than acetic acid.

A method described in the current patent application makes it possibleto isolate an alcohol-tolerant microorganism to produce a lower alkylalcohol from hydrogen gas, carbon dioxide gas, and/or carbon monoxidegas. In the described method, a mixed culture of microorganisms may beinoculated to media with a high concentration of at least one loweralkyl alcohol. The concentration of reactants and products is controlledto make production of the lower alkyl alcohol thermodynamicallyfeasible. The total gas pressure may be greater than 1 atmosphere. Inthis process, the microorganisms may be enriched by growing them underconditions that favor degradation of the alkyl alcohol, and thenconditions may be changed to select organisms that can also produce thedesired alkyl alcohol. The lower alkyl alcohol may be ethanol, propanol,or butanol or a combination thereof. The method can produce aconcentration of the alcohol greater than 6% by volume, or preferablygreater than 10% by volume, or greater.

A method is also described for producing at least one lower alkylalcohol from hydrogen gas, carbon dioxide gas and/or carbon monoxide gasin aqueous media, wherein the concentration of lower alkyl alcoholsincreases to greater than 4% by volume of the aqueous media. In thisprocess the lower alkyl alcohol can be ethanol, propanol or butanol, ora combination thereof. The alcohol or alcohols may be separated from theaqueous medium by distillation including vacuum distillation, which doesnot kill the microorganisms, or by distillation creating steam. Theproduction of alcohols may be created by incubation at a temperaturegreater than 35° C. or more preferably greater than 40° C. It can alsobe conducted at a higher temperature such as 55° C. or higher. The gaspressure may be greater than 1 atm, or more preferably be greater than 2atm or even more preferably greater than 3 or 4 atmospheres. The partialpressures of hydrogen gas and carbon dioxide gas can be adjusted to makeit thermodynamically more favorable to produce the desired alcohols overother products and over degradation of alcohols. The ratio of gases H₂to CO₂ may be greater than 1 or more preferably greater than 2. Evenmore preferably the ratio can be greater than 3. A ratio of 3 is optimalto produce a higher concentration of alcohol by volume, but a ratiogreater than 3 further favors alcohol production over correspondingcarboxylic acid production. With all of the conditions of partialpressures and concentrations of products, it may be thermodynamicallyfeasible to produce the alcohols to greater than 4% concentration of theaqueous media by volume. Volatile fatty acids may also be produced as aco-product and removed from the aqueous media. The volatile fatty acidsmay be removed by precipitating the conjugate base using addition of adivalent cation. For example, calcium or magnesium ions may be added byadding a salt or a base containing the ions. Precipitation may be aidedby increasing the pH.

This application also describes a method for producing one or morevolatile fatty acids from hydrogen gas, carbon dioxide gas and/or carbonmonoxide gas using at least one microorganism under conditions thatfavor synthesis of the desired volatile fatty acids over degradation.The concentration of the volatile fatty acids may be greater than 1%concentration by volume. Preferably, the concentration may exceed 2% or3% by volume. The volatile fatty acid may be acetic acid or acetate. Itmay also be a longer chain acid such as propionic acid or propionate,butyric acid or butyrate, iso-butyric acid or iso-butyrate, valeric acidor valerate, isovaleric acid or iso-valerate. It could also be lacticacid or lactate, or succinic acid or succinate. It could be alonger-chain carboxylic acid such as caproic acid, caprylic acid, orcapric acid. It could be a mixture of carboxylic acids or a singlespecies of carboxylic acid may comprise more than 50% of the totalcarboxylic acid produced by molar proportion. For example, it may bemore than 50% butyric acid by molar proportion. The pH of the fermentermay be adjusted to between 5 and 7 to increase production of carboxylicacids. The pressure in the fermenter may be greater than 1 atm or morepreferably may even be greater than 2 atm. With low concentrations ofCO₂, CO and H₂ or a skewed ratio of gases, the pressure may actuallyexceed several atmospheres in order to produce an adequate partialpressure of the gases to make it thermodynamically feasible to producethe desired carboxylic acids. The microorganisms used in the process maybe rumen microorganisms in pure culture or an undefined mixed culture.The one or more volatile fatty acids can be precipitated as theconjugate base using a divalent cation. For example, an ionic form ofcalcium or magnesium may be used, or a different cation. The pH of theaqueous medium may be increased to favor precipitation of the salt ofthe carboxylic acid. The rate of carboxylic acid production may begreater than 5 mM per liter per day. Preferably it may even be greaterthan 10 mM per liter per day. The carboxylic acid may be used as ananimal feed. Microbial protein produced in the process may also be usedas an animal feed.

Identification of an Example Microorganism

The phylogenetical relationship to other microorganisms was determinedfor one exemplary organism by base-pair sequence of the 16S ribosomalRNA. The isolate was selected as one of the early ones to produce a highconcentration of ethanol at a high rate from synthesis gases. Theorganism was purified an additional time using a streak plate and itsfermentation properties confirmed after the secondary purification. Anidentical colony was submitted for colony PCR and sequenced (Sequence IDNo. 1, below) on an automated sequencer. The sequence was comparedagainst a library and identified as having 99.89% homology (1 bp of 500differed) with Enterococcus avium. Greater than 97% homology was alsoobserved with E. gilvus (99.84), E. malodoratus (98.63), E. pseudoavium(98.21), and E. Raffinosus (97.89). Generally, greater than 97% homologyis commonly used as a benchmark for species identity of bacteria.

Having described the present invention, it will be apparent that changesand modifications may be made to the above-described embodiments withoutdeparting from the sprit and the scope of the present invention.

All volume or percentage of figures stated in the application are statedas volume/volume, unless stated otherwise or clearly contradicted by thecontext.

All references, including publications, patent applications, patents,and website content cited herein are hereby incorporated by reference tothe same extent as if each reference were individually and specificallyindicated to be incorporated by reference and was set forth in itsentirety herein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. The word “about,” when accompanying anumerical value, is to be construed as indicating a deviation of up toand inclusive of 10% from the stated numerical value. The use of any andall examples, or exemplary language (“e.g.” or “such as”) providedherein, is intended merely to better illuminate the invention and doesnot pose a limitation on the scope of the invention unless otherwiseclaimed. No language in the specification should be construed asindicating any non-claimed element as essential to the practice of theinvention.

What is claimed is:
 1. A process to develop a butanol-tolerantmicroorganism comprising a step in which a culture of microorganisms isgrown in a fermentation broth comprising at least 4% butanol by volume.2. The process of claim 1, wherein the fermentation broth comprises atleast 6% butanol by volume.
 3. The process of claim 1, wherein theculture of microorganisms is taken from an environmental source.
 4. Theprocess of claim 1, wherein the step in which a culture ofmicroorganisms is grown in a fermentation broth comprising at least 4%butanol by volume is used to screen for butanol-tolerant microorganisms.5. The process of claim 1, wherein the butanol-tolerant microorganism isa mutant microorganism that grows in a higher concentration of butanolthan microorganisms in the original culture.
 6. The process of claim 1,also comprising steps: diluting a culture of microorganisms untilindividual cells are separated from other cells in the culture; growingmultiple generations of the individual cells on agar medium; andphysically separating a colony that grows from an individual cell fromremaining microorganisms.
 7. The process of claim 1, wherein the processalso comprises a step in which exogenous hydrogen gas is infused into areactor vessel with the fermentation medium.
 8. The process of claim 1,wherein carbon dioxide and hydrogen gas partial pressures in a reactorvessel with the fermentation media make it thermodynamically infeasibleto degrade the butanol to carbon dioxide and hydrogen without anadditional source of energy.
 9. The process of claim 1, wherein the 4%butanol by volume is 1-butanol.
 10. A composition comprising thebutanol-tolerant microorganism developed in the process of claim
 1. 11.The composition of claim 10, wherein the butanol-tolerant microorganismgrows in a fermentation broth with at least 4% butanol by volume. 12.The composition of claim 10, wherein the butanol-tolerant microorganismcomprises genes that encode enzymes for producing butanol, and thebutanol-tolerant microorganism produces butanol.
 13. The composition ofclaim 10, wherein the butanol-tolerant microorganism grows in afermentation broth with at least 6% butanol by volume.
 14. Thecomposition of claim 10, wherein the butanol-tolerant microorganismconsumes glucose.
 15. The composition of claim 10, wherein thebutanol-tolerant microorganism produces butanol from cellobiose.
 16. Thecomposition of claim 10, wherein the butanol-tolerant microorganism isaerotolerant.
 17. A process to produce butanol comprising a step inwhich a carbon substrate is fermented within the composition of claim10.
 18. A process to produce butanol comprising steps: culturing abutanol-tolerant microorganism in a fermentation broth; wherein themicroorganism converts a substrate to the butanol; and whereinconcentration of butanol in the fermentation broth reaches at least 4%by volume
 19. The process of claim 18, wherein the concentration ofbutanol in the fermentation broth reaches at least 5% butanol by volume.20. The process of claim 18, wherein the concentration of butanol in thefermentation broth reaches at least 6% butanol by volume.
 21. Theprocess of claim 18, wherein the butanol is removed from thefermentation broth.
 22. The process of claim 18, wherein the substratecomprises glucose.
 23. The butanol produced from the process in claim18.
 24. A composition comprising a butanol-tolerant bacterium, whereinsaid butanol-tolerant bacterium tolerates at least 4% butanol by volume,grows under anaerobic conditions producing butanol, appears asgram-stain positive non-motile cocci, and comprises 16-S rRNA that is atleast 97% identical to a strain of Enterococcus.